Methods of isolation and use of cd39 stromal stem cells

ABSTRACT

Disclosed herein are methods of isolating SDC2+ stromal stem cells and exosomes from SDC2+ stromal stem cells by expression of surface marker CD39. Also disclosed herein are stromal stem cells and exosomes isolated by said methods.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.16/630,791, filed Jan. 13, 2020, which is a U.S. National Stage ofInternational Application No. PCT/IB2018/000939, filed Jul. 12, 2018,which claims the benefit of U.S. Provisional Application Nos.62/532,800, filed Jul. 14, 2017, and 62/534,631, filed Jul. 19, 2017,each of which is incorporated herein by reference in its entirety.

RELATED APPLICATIONS

This application incorporates by reference PCT Application No.PCT/IB2017/000091, filed on Jan. 13, 2017, in its entirety.

BACKGROUND

Mesenchymal stem cells (e.g., mesenchymal stromal stem cells and stromalcells) have been shown to have therapeutic value in treating a varietyof diseases. These cells have been found to associate with arterioles,sinusoidal endothelium and high endothelial vesicles in vivo, where theycan control endothelial cell activation and the trafficking of immunecells from the vasculature into target tissues. In addition mesenchymalstem cells have been shown to have immunosuppressive andanti-inflammatory properties such as avoiding allogeneic rejection andof inhibition immune cells, such as natural killer cells, neutrophils,dendritic cells, monocyte/macrophages and lymphocytes. Furthermore,mesenchymal stem cells have been found to produce immunosuppressingcytokines such as hepatocyte growth factor (HGF), IL-10, TGFβ1,cyclooxygenase 1 and 2, Syndecan-2 and PGE-2. The immunosuppressiveactivity of mesenchymal stem cells has been found to be increased in thepresence of inflammatory stimuli, specifically interferon-gamma. Theseproperties make mesenchymal stem cells and derivatives thereof (e.g.,mesenchymal stromal stem cells, stromal cells, and exosomes produced bymesenchymal stem cells) particularly intriguing in their potential totreat disease.

SUMMARY

Provided herein are methods of isolating a population of SDC2+ stromalstem cells from a mixed population of mammalian cells. Methods compriseone or more of the steps of (a) contacting the mixed population ofmammalian cells to a CD39 binding agent; (b) isolating cells bound tothe CD39 binding agent; and (c) measuring SDC2+ cell abundance in theisolated cells, thereby isolating the population of SDC2+ stromal stemcells. In some cases, the population of SDC2+ stromal stem cellscomprises a human, a mouse, a rat, or an equine cell. In some instances,a mixed population of mammalian cells is obtained from a source selectedfrom at least one of bone marrow, adipose tissue, skeletal muscle,endometrium, placenta, umbilical cord, Wharton's jelly, and cellsderived from pluripotent cells. A number of CD39 binding agents areconsistent with the disclosure herein. Often, the CD39 binding agentcomprises an antibody. In some cases, the antibody is raised to a CD39antigen. In some instances, the antibody comprises a variable domainthat specifically binds to at least one mammalian CD39 protein. Often,the antibody specifically binds to at least one of a human, a mouse, arat, and equine CD39 protein. In some cases, the antibody is conjugatedto a fluorophore. Sometimes, the antibody is conjugated to a bead.Often, at least 20% of the population of SDC2+ stromal stem cells isSDC2+. In some cases, at least 40% of the population of SDC2+ stromalstem cells is SDC2+. Often, at least 70% of the population of SDC2+stromal stem cells is SDC2+. In some cases, isolating the cells bound tothe CD39 binding agent comprises fluorescence activated cell sorting. Insome instances, isolating the cells bound to the CD39 binding agentcomprises magnetic-activated cell sorting. Often, the method comprisesculturing the isolated cells. In some cases, at least 90% of the SDC2+stromal stem cells are CD45−. In some cases, the population of SDC2+stromal stem cells further comprises CD25+ FoxP3+ regulatory T cells. Insome cases, the method comprises genetically modifying the cells tooverexpress an apyrase. In some cases, the method comprises geneticallymodifying the cells to overexpress CD39. In some cases, the methodcomprises genetically modifying the cells to overexpress CD39L3. In somecases, the method comprises genetically modifying the cells tooverexpress CD73.

Also provided herein are methods of preparing an immunomodulatorycomposition. Provided methods comprise a population of SDC2+ stromalstem cells, the method comprising (a) contacting a mixed population ofmammalian cells to a CD39 binding agent; (b) isolating cells bound tothe CD39 binding agent; and (c) measuring SDC2+ cell abundance in theisolated cells. In some cases, the population of SDC2+ stromal stemcells is a population of human, a mouse, a rat, or an equine cells.Often, the mixed population of mammalian cells are obtained from asource selected from at least one of bone marrow, adipose tissue,skeletal muscle, endometrium, placenta, umbilical cord, Wharton's jelly,and cells derived from pluripotent cells. In some cases, the CD39binding agent comprises an antibody. Often, the antibody is raised to aCD39 antigen. In some instances, the antibody comprises a variabledomain that specifically binds to at least one mammalian CD39 protein.In some cases, the antibody specifically binds to at least one of ahuman, a mouse, a rat, and equine CD39 protein. Often, the antibody isconjugated to a fluorophore. In some instances, the antibody isconjugated to a bead. In some cases, at least 20% of the population ofSDC2+ stromal stem cells is SDC2+. In some cases, at least 40% of thepopulation of SDC2+ stromal stem cells is SDC2+. In some cases, at least70% of the population of SDC2+ stromal stem cells is SDC2+. Often,isolating the cells bound to the CD39 binding agent comprisesfluorescence activated cell sorting. In some cases, the cells bound tothe CD39 binding agent comprises magnetic-activated cell sorting. Insome instances, culturing the cells isolated cells. Often, at least 90%of the SDC2+ stromal stem cells are CD45−. In some cases, thecomposition further comprises a buffer. In some instances, thecomposition further comprises an extracellular matrix. Often, theextracellular matrix is comprises a collagen. In some cases, theextracellular matrix is comprises a hyaluronic acid. Often, thecomposition further comprises a cryopreservant comprising DMSO. In someinstances, the composition further comprises a cryopreservant comprisingglycerol. In some cases, the population of SDC2+ stromal stem cellsfurther comprises CD25+ FoxP3+ regulatory T cells. In some cases, themethod comprises genetically modifying the cells to overexpress anapyrase. In some cases, the method comprises genetically modifying thecells to overexpress CD39. In some cases, the method comprisesgenetically modifying the cells to overexpress CD39L3. In some cases,the method comprises genetically modifying the cells to overexpressCD73.

Also provided herein are methods of isolating an immuno-modulatorycomposition. Some such methods comprise at least one of: (a) contactinga population of mammalian cells to a CD39 binding agent; (b) isolatingthe cells bound to the CD39 binding agent; and (c) recovering asupernatant comprising the exosomes from the isolated cells. In somecases, obtaining an exosome fraction from the supernatant. Often,obtaining an exosome fraction comprises centrifuging the supernatant. Insome instances, the centrifugation comprises centrifuging the cells atabout 100,000 g. In some cases, the centrifugation comprisescentrifuging the cells for at least one hour. In some instances, thecentrifugation comprises ultrafiltration. In some cases, thecentrifugation comprises size-exclusion liquid chromatography. Often,obtaining an exosome fraction comprises ultrafiltration. In some cases,obtaining an exosome fraction comprises size-exclusion liquidchromatography. In some instances, obtaining an exosome fractioncomprises contacting the supernatant to an antibody. Often, the antibodyis selected from at least one of an anti-CD39 antibody and an anti-SDC2antibody. In some cases, the exosomes are paracrine signaling exosomes.Often, the isolated cells are SDC2+. In some instances, the isolatedcells comprise mesenchymal stem cells. In some cases, at least 90% ofthe isolated cells are CD45−. In some instances, the method comprisesstoring the exosome fraction at room temperature. Often, the methodcomprises storing the exosome fraction without cryogenic preservation.In some cases, the method comprises adding an immunosuppressive drug tothe immuno-modulatory composition. Often, the isolated cells areperturbed to elicit exosome production. In some instances, the isolatedcells are cultured in a hollow-fiber bioreactor. In some cases, theisolated cells comprise CD25+ FoxP3+ regulatory T cells. In some cases,the method comprises genetically modifying the cells to overexpress anapyrase. In some cases, the method comprises genetically modifying thecells to overexpress CD39. In some cases, the method comprisesgenetically modifying the cells to overexpress CD39L3. In some cases,the method comprises genetically modifying the cells to overexpressCD73.

Also provided herein are methods of modulating an inflammation responsein a mammal. Some such methods comprise delivering a compositioncomprising SDC2+ cells to a site of the inflammation response, whereinthe SDC2+ cells are purified from a mixed population of cells byisolating cells based upon CD39 expression. In some instances, isolatingcells comprises collecting cells bound to a CD39 binding agent. Often,isolating the cells comprises culturing the cells bound to the CD39binding agent. In some cases, delivering comprises injecting thecomposition comprising SDC2+ cells. Often, delivering comprisestopically applying the composition comprising SDC2+ cells. In somecases, the composition comprises a hydrogel. In some instances, thecomposition comprises a collagen gel. In some cases, deliveringcomprises intraocularly administering the composition comprising SDC2+cells. Often, delivering comprises opthalamic application of thecomposition comprising SDC2+ cells. In some cases, delivering comprisesintravenous delivery the composition comprising SDC2+ cells. In someinstances, delivering comprises intra-lymph node injection of thecomposition comprising SDC2+ cells. In some cases, delivering comprisessubcutaneous delivery of the composition comprising SDC2+ cells. Often,delivering comprises intraperitoneal delivery of the compositioncomprising SDC2+ cells. In some cases, delivering comprises intrathecaldelivery of the composition comprising SDC2+ cells. In some instances,the SDC2+ cells wherein the SDC2+ cells are selected from at least oneof human, mouse, rat, and equine cells. In some cases, the mixedpopulation of mammalian cells are obtained from a source selected frombone marrow, adipose tissue, skelatal muscle, endometrium, placenta,umbilical cord, Wharton's jelly, and cells derived from pluripotentcells. Often, the CD39 binding agent comprises an antibody. In somecases, the antibody is raised to a CD39 antigen. In some instances, theantibody comprises a variable domain that specifically binds to at leastone mammalian CD39 protein. In some cases, the antibody specificallybinds to at least one of a human, a mouse, a rat, or equine CD39protein. In some cases, the antibody is conjugated to a fluorophore.Often, the antibody is conjugated to a bead. Often, at least 20% of theSDC2+ cells are SDC2+. In some cases, at least 40% of the SDC2+ cellsare SDC2+. In some instances, at least 70% of the SDC2+ cells are SDC2+.In some cases, isolating the cells comprises fluorescence activated cellsorting. In some cases, isolating the cells comprises magnetic-activatedcell sorting. Often, at least 90% of the SDC2+ cells are CD45−. In somecases, the method comprises delivering at least 10{circumflex over ( )}3SDC2+ cells. In some cases, the method comprises delivering at least10{circumflex over ( )}4 SDC2+ cells. In some cases, the methodcomprises delivering at least 10{circumflex over ( )}5 SDC2+ cells. Insome cases, the method comprises delivering at least 10{circumflex over( )}6 SDC2+ cells. In some cases, the population of SDC2+ stromal stemcells further comprises CD25+ FoxP3+ regulatory T cells. In some cases,the method comprises genetically modifying the cells to overexpress anapyrase. In some cases, the method comprises genetically modifying thecells to overexpress CD39. In some cases, the method comprisesgenetically modifying the cells to overexpress CD39L3. In some cases,the method comprises genetically modifying the cells to overexpressCD73. Often, the inflammation response comprises at least one of type 1diabetes, type 2 diabetes, sepsis, Crohn's disease, inflammatory bowelsyndrome, rheumatoid arthritis, graft versus host disease, multiplesclerosis, ALS, a dermal wound, a bone fracture, a cuncussion wound, aburn, atherosclerosis, nephropathy, cardiomyopathy, neuropathy, a kidneydisorder, kidney failure, a diabetic ulcer, a leg ulcer, ARDS, sepsis,inflammatory liver disease, myocarditis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, subacute bacterial endocarditis,anti-glomulerular basement membrane nephritis, interstitial cystitis,lupus enphritis, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, antisynthetase syndrome, asthma, chronicobstructive pulmonary disease, cystic fibrosis, atelectasis, bronchitis,emphasema, pneumonia, pulmonary endema, Alopecia Areata, autoimmuneAngioedema, autoimmune progesterone dermatitis, autoimmune urticaria,Bullous pemphigoid, Cicatricial pemphigoid, Dermatitis herpetiformis,Discoid lupus erythematosus, Epidermolysis bullosa acquisita, Erythemanodosum, Gestational pemphigoid, Hidradenitis suppurativa, Lichenplanus, Lichen sclerosus, Linear IgA disease, Morphea, Pemphigusvulgaris, Pityriasis lichenoides et varioliformis acuta, Mucha-Habermanndisease, Psoriasis, Systemic scleroderma, Vitiligo, Addison's disease,autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrometype 2, autoimmune polyendocrine syndrome type 3, autoimmunepancreatitis, autoimmune thyroiditis, Ord's thyroiditis, Graves'disease, Reproductive Organ disorder, autoimmune oophoritis,Endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmuneenteropathy, Celiac disease, Microscopic colitis, Ulcerative colitis,Antiphospholipid syndrome, Aplastic anemia, autoimmune hemolytic anemia,autoimmune lymphoproliferative syndrome, autoimmune neutropenia,autoimmune thrombocytopenic purpura, Cold agglutinin disease, Essentialmixed cryoglobulinemia, Evans syndrome, IgG4-related systemic disease,Paroxysmal nocturnal hemoglobinuria, Pernicious anemia, Pure red cellaplasia, Thrombocytopenia, Adiposis dolorosa, Adult-onset Still'sdisease, Ankylosing Spondylitis, CREST syndrome, Drug-induced lupus,Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome,Juvenile Arthritis, Lyme disease (Chronic), Mixed connective tissuedisease, Palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis,Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic LupusErythematosus, Undifferentiated connective tissue disease, cachexia,sarcophenia, Dermatomyositis, Fibromyalgia, Inclusion body myositis,Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellardegeneration, Polymyositis, Acute disseminated encephalomyelitis, Acutemotor axonal neuropathy, Anti-N-Methyl-D-Aspartate ReceptorEncephalitis, Balo concentric sclerosis, Bickerstaffs encephalitis,Chronic inflammatory demyelinating polyneuropathy, Guillain-Barrésyndrome, Hashimoto's encephalopathy, Idiopathic inflammatorydemyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiplesclerosis, Pediatric Autoimmune Neuropsychiatric Disorder Associatedwith Streptococcus, Progressive inflammatory neuropathy, Restless legsyndrome, Stiff person syndrome, Sydenham chorea, amyotrophic lateralsclerosis, Parkinson's disease, Alzheimer's disease, Transversemyelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome,Graves ophthalmopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, autoimmune inner ear disease, Ménière's disease,Anti-neutrophil cytoplasmic antibody-associated vasculitis, Behçet'sdisease, Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonleinpurpura, Kawasaki's disease, Leukocytoclastic vasculitis, Lupusvasculitis, Rheumatoid vasculitis, Microscopic polyangiitis,Polyarteritis nodosa, Polymyalgia rheumatica, Urticarial vasculitis, andVasculitis.

Also provided herein are methods of modulating an inflammation responsein a mammal. Some such methods comprise delivering a compositioncomprising exosomes to a site of the inflammation response, wherein theexosomes are SDC2+ and the exosomes are from SDC2+ cells isolated from amixed population of mammalian cells based upon CD39 expression. In somecases, isolating cells comprises collecting cells bound to a CD39binding agent. In some instances, isolating the cells comprisesculturing the cells bound to the CD39 binding agent. Often, deliveringcomprises injecting the composition comprising exosomes. Sometimes,delivering comprises topically applying the composition comprisingexosomes. In some cases, the composition comprises a hydrogel. Often,the composition comprises a collagen gel. In some instances, deliveringcomprises intraocularly administering the composition comprisingexosomes. In some cases, delivering comprises opthalamic application ofthe composition comprising exosomes. Sometimes, delivering comprisesintravenous delivery the composition comprising exosomes. In some cases,delivering comprises intra-lymph node injection of the compositioncomprising exosomes. In some cases, delivering comprises subcutaneousdelivery of the composition comprising exosomes. In some instances,delivering comprises intraperitoneal delivery of the compositioncomprising exosomes. In some cases, delivering comprises intrathecaldelivery of the composition comprising exosomes. Often, the exosomes areisolated from SDC2+ cells that are at least one of human, mouse, rat,and equine cells. Sometimes, the mixed population of mammalian cells areobtained from a source selected from bone marrow, adipose tissue,skelatal muscle, endometrium, placenta, umbilical cord, Wharton's jelly,and cells derived from pluripotent cells. In some cases, the CD39binding agent comprises an antibody. In some cases, the antibody israised to a CD39 antigen. In some instances, the antibody comprises avariable domain that specifically binds to at least one mammalian CD39protein. Often, the antibody specifically binds to at least one of ahuman, a mouse, a rat, or equine CD39 protein. In some cases, theantibody is conjugated to a fluorophore. Sometimes, the antibody isconjugated to a bead. In some cases, at least 20% of the exosomes areSDC2+. In some cases, at least 40% of the exosomes are SDC2+. In somecases, at least 70% of the exosomes are SDC2+. Often, isolating thecells comprises fluorescence activated cell sorting. In some cases,isolating the cells comprises magnetic-activated cell sorting. In someinstances, the method further comprises culturing the cells bound to theCD39 binding agent. In some cases, the cells are CD45−. In some cases,the method comprises delivering at least 10{circumflex over ( )}6exosomes. In some cases, the method comprises delivering at least10{circumflex over ( )}7 exosomes. In some cases, the method comprisesdelivering at least 10{circumflex over ( )}8 exosomes. In some cases,the method comprises delivering at least 10{circumflex over ( )}9exosomes. In some cases, the population of SDC2+ stromal stem cellsfurther comprises CD25+ FoxP3+ regulatory T cells. In some cases, themethod comprises genetically modifying the cells to overexpress anapyrase. In some cases, the method comprises genetically modifying thecells to overexpress CD39. In some cases, the method comprisesgenetically modifying the cells to overexpress CD39L3. In some cases,the method comprises genetically modifying the cells to overexpressCD73. Often, the inflammation response comprises at least one of type 1diabetes, type 2 diabetes, sepsis, Crohn's disease, inflammatory bowelsyndrome, rheumatoid arthritis, graft versus host disease, multiplesclerosis, ALS, a dermal wound, a bone fracture, a concussion wound, aburn, atherosclerosis, nephropathy, cardiomyopathy, neuropathy, a kidneydisorder, kidney failure, a diabetic ulcer, a leg ulcer, ARDS, sepsis,inflammatory liver disease, myocarditis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, subacute bacterial endocarditis,anti-glomulerular basement membrane nephritis, interstitial cystitis,lupus enphritis, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, antisynthetase syndrome, asthma, chronicobstructive pulmonary disease, cystic fibrosis, atelectasis, bronchitis,emphasema, pneumonia, pulmonary edema, Alopecia Areata, autoimmuneAngioedema, autoimmune progesterone dermatitis, autoimmune urticaria,Bullous pemphigoid, Cicatricial pemphigoid, Dermatitis herpetiformis,Discoid lupus erythematosus, Epidermolysis bullosa acquisita, Erythemanodosum, Gestational pemphigoid, Hidradenitis suppurativa, Lichenplanus, Lichen sclerosus, Linear IgA disease, Morphea, Pemphigusvulgaris, Pityriasis lichenoides et varioliformis acuta, Mucha-Habermanndisease, Psoriasis, Systemic scleroderma, Vitiligo, Addison's disease,autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrometype 2, autoimmune polyendocrine syndrome type 3, autoimmunepancreatitis, autoimmune thyroiditis, Ord's thyroiditis, Graves'disease, Reproductive Organ disorder, autoimmune oophoritis,Endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmuneenteropathy, Celiac disease, Microscopic colitis, Ulcerative colitis,Antiphospholipid syndrome, Aplastic anemia, autoimmune hemolytic anemia,autoimmune lymphoproliferative syndrome, autoimmune neutropenia,autoimmune thrombocytopenic purpura, Cold agglutinin disease, Essentialmixed cryoglobulinemia, Evans syndrome, IgG4-related systemic disease,Paroxysmal nocturnal hemoglobinuria, Pernicious anemia, Pure red cellaplasia, Thrombocytopenia, Adiposis dolorosa, Adult-onset Still'sdisease, Ankylosing Spondylitis, CREST syndrome, Drug-induced lupus,Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome,Juvenile Arthritis, Lyme disease (Chronic), Mixed connective tissuedisease, Palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis,Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic LupusErythematosus, Undifferentiated connective tissue disease, cachexia,sarcophenia, Dermatomyositis, Fibromyalgia, Inclusion body myositis,Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellardegeneration, Polymyositis, Acute disseminated encephalomyelitis, Acutemotor axonal neuropathy, Anti-N-Methyl-D-Aspartate ReceptorEncephalitis, Balo concentric sclerosis, Bickerstaffs encephalitis,Chronic inflammatory demyelinating polyneuropathy, Guillain-Barrésyndrome, Hashimoto's encephalopathy, Idiopathic inflammatorydemyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiplesclerosis, Pediatric Autoimmune Neuropsychiatric Disorder Associatedwith Streptococcus, Progressive inflammatory neuropathy, Restless legsyndrome, Stiff person syndrome, Sydenham chorea, amyotrophic lateralsclerosis, Parkinson's disease, Alzheimer's disease, Transversemyelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome,Graves ophthalmopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, autoimmune inner ear disease, Meniere's disease,Anti-neutrophil cytoplasmic antibody-associated vasculitis, Behçet'sdisease, Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonleinpurpura, Kawasaki's disease, Leukocytoclastic vasculitis, Lupusvasculitis, Rheumatoid vasculitis, Microscopic polyangiitis,Polyarteritis nodosa, Polymyalgia rheumatica, Urticarial vasculitis, andVasculitis.

Also provided herein are compositions comprising a population of cellsthat is at least 20% SDC2+, wherein the population of cells is bound toa CD39 binding agent. In some cases, the population of cells is at least30% SDC2+. In some cases, the population of cells is at least 40% SDC2+.In some cases, the population of cells is at least 50% SDC2+. In somecases, the population of cells is at least 60% SDC2+. In some cases, thepopulation of cells is at least 70% SDC2+. In some cases, the populationof cells is at least 80% SDC2+. In some cases, the population of cellsis at least 90% SDC2+. In some cases, the population of cells is atleast 95% SDC2+. In some cases, the population of cells is at least 99%SDC2+. In some cases, the population of cells is at least 20% CD39+. Insome cases, the population of cells is at least 30% CD39+. In somecases, the population of cells is at least 40% CD39+. In some cases, thepopulation of cells is at least 50% CD39+. In some cases, the populationof cells is at least 60% CD39+. In some cases, the population of cellsis at least 70% CD39+. In some cases, the population of cells is atleast 80% CD39+. In some cases, the population of cells is at least 90%CD39+. In some cases, the population of cells is at least 95% CD39+. Insome cases, the population of cells is at least 99% CD39+. In somecases, at least 90% of the population of cells is CD45−. In someinstances, the composition further comprises at least 10{circumflex over( )}6 exosomes isolated from a population of cells bound to a CD39binding agent. In some instances, the composition further comprises atleast 10{circumflex over ( )}7 exosomes isolated from a population ofcells bound to a CD39 binding agent. In some instances, the compositionfurther comprises at least 10{circumflex over ( )}8 exosomes isolatedfrom a population of cells bound to a CD39 binding agent. In someinstances, the composition further comprises at least 1 μg of exosomesisolated from a population of cells bound to a CD39 binding agent. Insome instances, the composition further comprises at least 10 μg ofexosomes isolated from a population of cells bound to a CD39 bindingagent. In some instances, the composition further comprises at least 20μg of exosomes isolated from a population of cells bound to a CD39binding agent. In some instances, the composition further comprises atleast 50 μg of exosomes isolated from a population of cells bound to aCD39 binding agent. In some instances, the composition further comprisesat least 100 μg of exosomes isolated from a population of cells bound toa CD39 binding agent. In some instances, the composition furthercomprises at least 150 μg of exosomes isolated from a population ofcells bound to a CD39 binding agent. In some instances, the compositionfurther comprises at least 200 μg of exosomes isolated from a populationof cells bound to a CD39 binding agent. In some instances, thecomposition further comprises at least 250 μg of exosomes isolated froma population of cells bound to a CD39 binding agent. In some instances,the composition further comprises at least 500 μg of exosomes isolatedfrom a population of cells bound to a CD39 binding agent. In someinstances, the composition further comprises at least 750 μg of exosomesisolated from a population of cells bound to a CD39 binding agent. Insome instances, the composition further comprises at least 1000 μg ofexosomes isolated from a population of cells bound to a CD39 bindingagent. In some cases, the CD39 binding agent comprises an antibody. Insome cases, the antibody is raised to a CD39 antigen. In some instances,the antibody comprises a variable domain that specifically binds to atleast one mammalian CD39 protein. Often, the antibody specifically bindsto at least one of a human, a mouse, a rat, or equine CD39 protein. Insome cases, the antibody is conjugated to a fluorophore. Sometimes, theantibody is conjugated to a bead. In some instances, the compositionfurther comprises a buffer. In some instances, the composition furthercomprises a cyropreservant comprising DMSO. In some instances, thecomposition further comprises a cyropreservant comprising glycerol. Insome cases, the composition comprises at least 10{circumflex over ( )}3cells. In some cases, the composition comprises at least 10{circumflexover ( )}4 cells. In some cases, the composition comprises at least10{circumflex over ( )}5 cells. In some cases, the composition comprisesat least 10{circumflex over ( )}6 cells. In some cases, the compositioncomprises at least 10{circumflex over ( )}7 cells. In some cases, thepopulation of cells comprises CD25+ FoxP3+ regulatory T cells. In somecases, the population of cells is genetically modified to overexpress anapyrase. In some cases, the population of cells is genetically modifiedto overexpress CD39. In some cases, the population of cells isgenetically modified to overexpress CD39L3. In some cases, thepopulation of cells is genetically modified to overexpress CD73.

Also provided herein are compositions comprising exosomes, wherein theexosomes are at least 20% SDC2+ and wherein the exosomes are bound to aCD39 binding agent. In some cases, the exosomes are at least 30% SDC2+.In some cases, the exosomes are at least 40% SDC2+. In some cases, theexosomes are at least 50% SDC2+. In some cases, the exosomes are atleast 60% SDC2+. In some cases, the exosomes are at least 70% SDC2+. Insome cases, the exosomes are at least 80% SDC2+. In some cases, theexosomes are at least 90% SDC2+. In some cases, the exosomes are atleast 95% SDC2+. In some cases, the exosomes are at least 99% SDC2+. Insome cases, the exosomes are at least 20% CD39+. In some cases, theexosomes are at least 30% CD39+. In some cases, the exosomes are atleast 40% CD39+. In some cases, the exosomes are at least 50% CD39+. Insome cases, the exosomes are at least 60% CD39+. In some cases, theexosomes are at least 70% CD39+. In some cases, the exosomes are atleast 80% CD39+. In some cases, the exosomes are at least 90% CD39+. Insome cases, the exosomes are at least 95% CD39+. In some cases, theexosomes are at least 99% CD39+. In some cases, the population is CD45−.In some instances, the composition comprises at least 10{circumflex over( )}6 exosomes. In some instances, the composition comprises at least10{circumflex over ( )}7 exosomes. In some instances, the compositioncomprises at least 10{circumflex over ( )}8 exosomes. In some instances,the composition comprises at least 1 mg of exosomes. In some instances,the composition comprises at least 10 mg of exosomes. In some instances,the composition comprises at least 20 mg of exosomes. In some instances,the composition comprises at least 50 mg of exosomes. In some instances,the composition comprises at least 100 mg of exosomes. In someinstances, the composition comprises at least 150 mg of exosomes. Insome instances, the composition comprises at least 200 mg of exosomes.In some instances, the composition comprises at least 250 mg ofexosomes. In some instances, the composition comprises at least 500 mgof exosomes. In some instances, the composition comprises at least 750mg of exosomes. In some instances, the composition comprises at least1000 mg of exosomes. In some instances, the composition comprises abuffer. In some cases, the CD39 binding agent comprises an antibody. Insome cases, the antibody is raised to a CD39 antigen. In some instances,the antibody comprises a variable domain that specifically binds to atleast one mammalian CD39 protein. Often, the antibody specifically bindsto at least one of a human, a mouse, a rat, or equine CD39 protein. Insome cases, the antibody is conjugated to a fluorophore. Sometimes, theantibody is conjugated to a bead.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe specification and appended claims. Further understanding of thefeatures and advantages of the present invention will be obtained byreference to the following detailed description that sets forthillustrative embodiments, in which the principles of the invention areutilized, and the accompanying drawings of which:

FIG. 1A shows an exemplary gating strategy for isolating stromal stemcells from umbilical cord.

FIG. 1B shows an exemplary analysis of CD39 and CD362 (SDC2) expressionin the studied umbilical cord cell population. Double positive cells arehighlighted with a bolded box and an arrow.

FIG. 1C shows relative population percentage of CD362+ versusCD39+/CD362+ cells.

FIG. 1D shows an exemplary colony formation assay on CD362+/CD39+ cells.

FIG. 1E shows an exemplary colony formation assay on unsorted (wildtype)cells.

FIG. 2A shows a representative gating control and identification ofCD39+/CD362+ cells from bone marrow (highlighted by bold square).

FIG. 2B shows relative population percentage of CD39+/CD362+ cellsversus CD39hi/CD362+ cells.

FIG. 2C shows tabular results of colony formation assay for unsortedcells, CD39−/CD362+ cells, CD39+/CD362+ cells, CD39+/CD362− cells, anddouble negative cells.

FIG. 3A shows a representative Miltenyi MACSQuant Tyto sort gate andanalysis for identification of CD39+/CD362+ cells from human bone marrow(highlighted in bold squares).

FIG. 3B shows tabular results of colony formation assay for unsortedcells, CD39−/CD362+ cells, CD39+/CD362+ cells, CD39hi/CD362+,CD39+/CD362− cells, and double negative cells using both the MiltenyiMACSQuant Tyto sort and BD FACSAria cell sorters from human bone marrow.

FIG. 4 illustrates exosome synthesis, release and uptake by an adjacentcell.

FIG. 5 shows SDC2 expression by exosomes derived from SDC2+ mesenchymalstromal cells (MSC) by transmission electron microscopy.

FIG. 6 shows SDC2 expression by exosomes derived from SDC2+ mesenchymalstromal cells (MSC) by Western blot.

FIG. 7 shows arterial FI 0.3 in rats treated with PBS vehicle, humanmesenchymal stem cells, or exosomes in a rat model of ARDS.

FIG. 8 shows arterial FI 1.0 in rats treated with PBS vehicle, humanmesenchymal stem cells, or exosomes in a rat model of ARDS.

FIG. 9 shows arterial FI 0.3 in rats treated with PBS vehicle, bonemarrow (BM) exosomes, or human umbilical cord (HUC) exosomes in a ratmodel of ARDS.

FIG. 10 shows arterial FI 1.0 in rats treated with PBS vehicle, bonemarrow (BM) exosomes, or human umbilical cord (HUC) exosomes in a ratmodel of ARDS.

FIG. 11 shows bronchioaveolar lavage (BAL) bacterial load in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS.

FIG. 12 shows bronchioaveolar lavage (BAL) total cell count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS.

FIG. 13 shows bronchioaveolar lavage (BAL) neutrophil count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS.

FIG. 14 shows static lung compliance in rats treated with PBS vehicle,human mesenchymal stem cells, or exosomes in a rat model of ARDS.

FIG. 15 shows lung wet dry ratio in rats treated with PBS vehicle, humanmesenchymal stem cells, or exosomes in a rat model of ARDS.

FIG. 16 shows results from a scratch assay with A549 cells treated withserum free media or exosomes for 48 hours.

FIG. 17 shows results a scratch assay with A549 cells, plate 1.

FIG. 18 shows a bar graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultrafiltration.

FIG. 19 shows a scatter graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultrafiltration.

FIG. 20 shows a bar graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultracentrifugation.

FIG. 21 shows a scatter graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultracentrifugation.

FIG. 22 shows arterial FI 0.3 in rats treated with PBS vehicle, humanmesenchymal stem cells (10 mill/kg), or exosomes (200 μg, IV) in a ratmodel of ARDS.

FIG. 23 shows arterial FI 1.0 in rats treated with PBS vehicle, humanmesenchymal stem cells (10 mill/kg), or exosomes (200 μg, IV) in a ratmodel of ARDS.

FIG. 24 shows static lung compliance in rats treated with PBS vehicle,human mesenchymal stem cells (10 mill/kg), or exosomes (200 μg, IV) in arat model of ARDS.

FIG. 25 shows bronchioaveolar lavage (BAL) bacterial load in ratstreated with PBS vehicle, human mesenchymal stem cells (10 mill/kg), orexosomes (200 μg, IV) in a rat model of ARDS.

FIG. 26 shows bronchioaveolar lavage (BAL) total cell count in ratstreated with PBS vehicle, human mesenchymal stem cells (10 mill/kg), orexosomes (200 μg, IV) in a rat model of ARDS.

FIG. 27 shows bronchioaveolar lavage (BAL) neutrophil count in ratstreated with PBS vehicle, human mesenchymal stem cells (10 mill/kg), orexosomes (200 mg, IV) in a rat model of ARDS.

DETAILED DESCRIPTION OF THE INVENTION

Stromal stem cells or mesenchymal stromal cells have shown therapeuticvalue, for example, in their ability to migrate to the site of tissueinjury and relative ease of in vitro expansion. In particular SDC2+stromal stem cells have shown efficacy in treatment of a wide range ofdiseases. Alternative methods of preparing SDC2+ stromal stem cells aredisclosed herein, wherein isolation of SDC2+ stromal stem cells is basedon expression of CD39.

CD39 and CD39L3 are apyrase enzymes. Apyrase enzymes cleave phosphategroups from ATP and ADP to yield AMP and phosphate. More generallyspeaking, apyrase enzymes are extracellular nucleosidases, which mediatecatabolism of extracellular nucleotides, such as ATP and ADP. CD39 andCD39L3 cell surface expression often correlates with SDC2+ cell surfaceexpression, such that CD39 or CD39L3 is effective as a marker cellsurface protein for isolation of SDC2+ stromal stem cells. Extracellularnucleosideases, in some cases, mediate catabolism of extracellular ATPand ADP to AMP, which is further degraded to adenosine. CD73 oftendegrades AMP to adenosine. Additional examples of extracellularnucleosidases include CD39L1, CD39L2, and CD39L4. As ATP and ADP actantagonistically to free adenosine outside of the cell to mediate anumber of responses including cell differentiation and inflammation,CD39, CD39 homologues, and even structurally diverse, functionallyanalogous enzymes may have a common impact on extracellular ATPsignaling.

In some cases, apyrases, such as extracellular nucleosidases, areattached to a cell via a transmembrane domain. In some cases,extracellular nucleosidases are soluble proteins outside the cell.Apyrase activity is effected through full length proteins or,alternately, through proteins having an apyrase extracellular domain oran extracellular domain sharing sequence, structural similarity orfunctional similarity to an apyrase, so as to commonly modulate ATP orADP concentration relative to adenosine or extracellular purineconcentration.

SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions and methods of isolation based on CD39 expression and usesfor such cells in modulation the inflammatory response are disclosedherein. In some cases these compositions mediate of paracrine signalingor deliver paracrine signaling components.

Additional uses for SDC2+ and SDC2+CD39 stromal stem cell and SDC2+ andSDC2+CD39+ exosome compositions include compositions that includeanti-inflammatory therapeutics formulated with the exosomes. Thisfeature would allow a therapeutic to be delivered to an individual whoneeds treatment. For example, an anti-TNF antibody, such as infliximab,could be formulated with the SDC2+ and SDC2+CD39+ stromal stem cell andSDC2+ and SDC2+CD39+ exosome compositions. Alternately or on combinationa native signaling component, such as a paracrine signaling component,is contained in some SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+and SDC2+CD39+ exosome compositions.

In addition, SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ andSDC2+CD39+ exosome compositions, such as those described herein, canalso be combined with regulatory T cells, such as CD4+CD25+FOXP3+regulatory T cells, to create therapeutics that treat inflammatory orimmune diseases. The exosome compositions, in this case, would enhancethe activity, potency and longevity of a regulatory T cell therapeutic.

Methods of Isolation of SDC2+ Stromal Stem Cells Using CD39 BindingAgents

SDC2+ stromal stem cells disclosed herein are isolated or purified basedon expression of CD39. In some cases, such methods isolate SDC2+ stromalstem cells using a CD39 binding agent. The method of isolation orpurification involves isolating stromal stem cells from a population ofmammalian cells. In some cases, methods of isolation or purification ofSDC2+ stromal stem cell compositions comprise obtaining a population ofmammalian cells for isolation of the SDC2+ stromal stem cells,contacting the population of mammalian cells with a CD39 binding agent,recovering cells bound to the CD39 binding agent, and thereby obtainingSDC2+ stromal stem cells. The method of isolation or purificationresults in a stromal stem cell composition enriched for SDC2+ cells.Some such cell populations comprise SDC2+ stromal stem cells, wherein atleast 20% of the stromal stem cells comprise express SDC2+. In somecases, the CD39 binding agent comprises an antibody or an antibodyfragment. In some cases, the antibody is raised to a CD39 antigen. Insome instances, the antibody comprises a variable domain thatspecifically binds to at least one mammalian CD39 protein. Often, theantibody specifically binds to at least one of a human, a mouse, a rat,or equine CD39 protein. In some cases, the antibody is conjugated to afluorophore. Sometimes, the antibody is conjugated to a bead.

Isolation or purification of SDC2+ stromal stem cells is accomplishedwith the use of a CD39 binding agent. In some cases, the CD39 bindingagent comprises one or more antibodies to CD39 or fragments thereof.Some methods of isolation or purification comprise incubating apopulation of mammalian cells with a CD39 binding agent and retainingonly the cells bound to the CD39 binding agent. CD39 binding agents,such as CD39 antibodies suitable for methods disclosed herein have theproperty of binding specifically to CD39. Incubation of a population ofmammalian cells with the CD39 binding agent is done in a buffer thatpromotes specific binding of the CD39 binding agent to CD39 and at atemperature that facilitates antibody binding and cell viability andstability. In some cases, the incubation is done at room temperature. Insome cases, the incubation is done at 4° C. An incubation buffer oftencomprises at least one or a buffer, a detergent, and a salt. Alternatelyor in combination, the CD39 binding agent comprises a binding agent thatis not an antibody, such as a CD39 receptor or an oligomer that bindsCD39. Isolated or purified SDC2+ stromal stem cells are often preparedin a buffer or excipient suitable for storage or for administration toan individual in need thereof.

Stromal cells for methods of isolation or purification of SDC2+ stromalstem cells, in some cases, are modified to increase CD39, CD39L3, orother apyrase protein expression. It is observed that suchoverexpression improves the therapeutic efficacy of the stromal stemcells resulting from the isolation or purification method. In somecases, cells are genetically modified to overexpress CD39. In somecases, cells are genetically modified to overexpress CD39L3.Alternately, overexpression of an apyrase, apyrase functional fragment,apyrase extracellular domain, or extracellular domain functionalfragment, or protein having an apyrase extracellular domain activity, issufficient to effect improved therapeutic efficacy.

Genetic modification of stromal cells is accomplished by methodsincluding but not limited to transfection of the stromal cells with oneor more plasmids that comprise the CD39 or CD39L3 coding sequence and apromoter, such as a CMV, SV40, EF1a, or CAG promoter. Geneticmodification of stromal cells, in some cases, is accomplished byinfection of the stromal cells with a virus that comprises the CD39 orCD39L3 coding sequence and a promoter.

Methods of isolation or purification of SDC2+ stromal stem cells using aCD39 binding agent yield compositions comprising a specific proportionof SDC2+ stromal stem cells. In some cases, the proportion of stromalstem cells in the composition purified using a CD39 binding agent thatcomprise SDC2 is within a range of 20% to 99%. Some methods of isolationor purification result in a stromal stem cell composition where theproportion of stromal stem cells in the composition that comprise SDC2is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, orgreater than 99%. In some cases, method of isolation or purificationresults in a stromal stem cell composition where the proportion ofstromal stem cells in the composition that comprise CD39 is at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, or greaterthan 99%.

Methods of isolation or purification of cells binding to a CD39 bindingagent, such as isolation or purification of SDC2+ stromal stem cellsusing a CD39 binding agent such as an antibody or CD39 conjugate, insome cases, yield compositions comprising SDC2+ cells and furthercomprising regulatory T cells. In some cases, the regulatory T cells areCD25+. In some cases, the regulatory T cells are FoxP3+. In some cases,the regulatory T cells are CD25+ and FoxP3+.

Disclosed herein are methods of isolation or purification of SDC2+stromal stem cells binding to a CD39 binding agent in a therapeuticallyeffective amount. A minimum therapeutically effective amount of SDC2+stromal stem cells purified using a CD39 binding agent, in some cases,ranges from 10{circumflex over ( )}3-10{circumflex over ( )}8 cells, forexample, 10{circumflex over ( )}3, 10{circumflex over ( )}4,10{circumflex over ( )}5, 10{circumflex over ( )}6, 10{circumflex over( )}7, 10{circumflex over ( )}8, or more than 10{circumflex over ( )}8cells. Sometimes substantially fewer or substantially more cellsconstitute a therapeutic amount. Isolation or purification of SDC2+stromal stem cells via CD39 binding agents, in some cases, comprisesconcentrating the SDC2+ stromal stem cell composition to be diluted in abuffer or excipient by the individual prior to administration. In somecases, isolation or purification of SDC2+ stromal stem cell compositionsbinding to a CD39 binding agent comprises diluting the SDC2+ stromalstem cell composition in a buffer or excipient so as to make it ready tobe administered to the individual. In some cases, methods of isolationor purification of SDC2+ stromal stem cell compositions binding to aCD39 binding agent results in single use vials or IV bags. In somecases, methods of isolation or purification of SDC2+ stromal stem cellcompositions using CD39 binding agents results in multiple doses arepresent in a single container.

Methods of isolation or purification of SDC2+ stromal stem cells using abinding agent such as an anti-CD39 antibody comprises isolating the CD39antibody-SDC2+ stromal stem cell complex from the population ofmammalian cells. CD39 antibody-SDC2+ stromal stem cell complexes arepurified any number of suitable methods including but not limited tofluorescence activated cell sorting (FACS), immunoprecipitation, columnpurification using protein A beads, column purification using protein Gbeads, column purification using biotinylated beads and a biotinylatedsecondary antibody, and magnetic bead based separation methods. SDC2+stromal stem cells are then eluted from the antibody using a bufferedsalt solution having stringency sufficient to elute the SDC2+ stromalstem cells from the antibody. Buffered salt solutions are removed fromthe exosome composition using a centrifugation or dilution procedure.The resulting isolated, purified SDC2+ stromal stem cells are thendiluted in a physiologically acceptable buffer or excipient and frozenor otherwise stored at a temperature where the SDC2+ stromal stem cellswith retain potency and stability. Optionally, the SDC2+ stromal stemcells are cultured in a mammalian cell culture buffer to expand the cellnumber before preparing the cells for administration.

Methods of Isolation of SDC2+ Exosomes

Disclosed herein are methods of isolating, purifying or enriching forSDC2+ exosomes using CD39 binding agents. Exosome compositions such asparacrine signaling exosome compositions disclosed herein are isolatedor purified. The method of isolation or purification involves isolatingexosomes from a population of stromal cells such as SDC2+ stromal cells,isolated using a CD39 binding agent by methods provided herein. Methodsof isolation or purification of exosome compositions often compriseobtaining a cell population enriched for SDC2+ cells from a mixedpopulation of mammalian cells using a CD39 binding agent for isolationof the exosomes, recovering a supernatant from said cell population, andobtaining an exosome fraction from the supernatant. Exosomecompositions, such as paracrine signaling exosome compositions disclosedherein, in some cases, express SDC2. Often, exosome compositions hereinexpress CD39. In some cases, the SDC2 is found in the interior of theexosome. In some cases, the SDC2 is found on the exterior of theexosome. CD39 is alternatively found in the interior of the exosome oron the exterior of the exosome. The method of isolation or purificationresults in an exosome composition comprising exosomes, wherein at least20% of the exosomes comprise SDC2.

Stromal cells for methods of isolation or purification of exosomecompositions such as paracrine signaling exosome compositions, in somecases, are modified to increase the yield of exosome compositionsresulting from the isolation or purification method. In some cases,cells are genetically modified to overexpress SDC2. In some cases, cellsare genetically modified to overexpress CD39. Genetic modification ofstromal cells is accomplished by methods including but not limited totransfection of the stromal cells with one or more plasmids thatcomprise the SDC2 or CD39 coding sequence and a promoter, such as a CMV,SV40, EF1a, or CAG promoter. Genetic modification of stromal cells, insome cases, is accomplished by infection of the stromal cells with avirus that comprises the SDC2 or CD39 coding sequence and a promoter.

Stromal cells for methods of isolation or purification of exosomecompositions using CD39 binding agents provided herein, such asparacrine signaling exosome compositions, are genetically modified tooverexpress a protein associated with controlling the cytoskeleton. Insome cases, cells are genetically modified to overexpress cortactin.Genetic modification of stromal cells is accomplished by methodsincluding but not limited to transfection of the stromal cells with oneor more plasmids that comprise the cortactin coding sequence and apromoter, such as a CMV, SV40, EF1a, or CAG promoter. Geneticmodification of stromal cells, in some cases, is accomplished byinfection of the stromal cells with a virus that comprises the cortactincoding sequence and a promoter.

Often, stromal cells for methods of isolation or purification of exosomecompositions using CD39 binding agents such as paracrine signalingexosome compositions are irradiated to increase the yield of exosomecompositions resulting from the isolation of purification method.Irradiation of cells includes but is not limited to exposing the stromalcells to a source of radiation, such as an alpha radiation source, abeta radiation source, or a gamma radiation source. In some cases,stromal cells are irradiated using gamma-irradiation.

Alternatively or in combination, stromal cells for methods of isolationor purification of exosome compositions using CD39 binding agents suchas paracrine signaling exosome composition are subjected to aninflammatory stimulus. Inflammatory stimuli include but are not limitedto TNF-alpha, interferon-gamma, interferon-beta, interleukin-1b, TLRagonists, Poly I:C, and LPS.

Additionally, stromal cells for methods of isolation or purification ofexosome compositions using CD39 binding agents are subjected to a growtharrest. Growth arrest is understood by those of skill in the art asslowing or stopping division of cells. Methods of growth arrest includebut are not limited to irradiation, mitomycin-c, TGFb stimulation, andgrowing cells to confluence.

Methods of isolation or purification of exosome compositions using CD39binding agents, such as paracrine signaling exosome compositions yieldcompositions comprising a specific proportion of exosomes that compriseSDC2. In some cases, the proportion of exosomes in the composition thatcomprise SDC2 is within a range of 20% to 99%. In some cases, method ofisolation or purification results in an exosome composition where theproportion of exosomes in the composition that comprise SDC2 is at least20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater. In somecases, the proportion of exosomes in the composition that comprise CD39is within a range of 20% to 99%. In some cases, method of isolation orpurification results in an exosome composition where the proportion ofexosomes in the composition that comprise CD39 is at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater.

Some methods of isolation or purification of exosome compositions usingCD39 binding agents, such as paracrine signaling exosome compositionsuse continuous production technology, such as hollow-fiber bioreactortechnology. Hollow-fiber bioreactors (HFBRs) have high surface-to-volumeratios which support large numbers of cells at high densities. ExemplaryHFBRs include but are not limited to FiberCell Systems and TerumoQuantum Cell Expansion System. Such systems can support from about10{circumflex over ( )}7 cells to about 10{circumflex over ( )}8 cells,although cell amounts outside this range may also be employed in somecases. Advantages of HFBRs include but are not limited to a fiber with amolecular weight cutoff of 5-20 kDa which allows nutrients and wasteproducts to pass through but exosomes are retained in the reactor andconcentrated up to 100 times. Cells bound to the support do not requiresplitting as cell lines can grow to post-confluence without significantapoptosis. In some cases, collection of exosomes is maintained overseveral months of continuous production. All of these factors combine toallow exosomes to be secreted in large numbers and concentratedsignificantly in the small volume of the extracapillary space of thecartridge. Exosomes cannot cross the fiber in either direction so cellculture serum can be used in the circulating medium withoutcontaminating the secreted exosomes within the extracapillary space ofthe cartridge.

Methods of isolation or purification of exosome compositions using CD39binding agents, such as paracrine signaling exosome compositions resultin an exosome composition that is free of living cells, in other words,the resulting exosome composition does not comprise a living cell.Isolation or purification of exosome compositions results in acomposition that is non-tumorigenic. That is, the exosome compositionobtained by methods of isolation or purification disclosed herein doesnot cause tumors or cancer to develop in a mammal that has been treatedwith or given one or more doses of the exosome composition.

Methods of isolation or purification using CD39 binding agents resultdisclosed herein result in a stable composition. For example, theexosome composition such as paracrine signaling exosome compositionobtained by the methods of isolation or purification is stable at roomtemperature (20 to 25° C.), at cold temperatures (3 to 5° C.), orfreezing temperatures (−150 to 0° C.). In some cases, stability isimproved by the addition of suitable buffers or excipients. Non-limitingexamples of excipients include sucrose, trehalose, polyethylene glycol,a polysaccharide, a carrier protein, an inert protein, dextran, hydroxylethyl starch (HETA), PEG-4000, gelatin, PLGA, Eudragit RS 100Nanoparticles, and combinations thereof.

Methods of isolation or purification of exosome compositions using CD39binding agents disclosed herein result in an exosome composition thatcan retain potency or activity after being frozen or cryopreservedwithout the use of a cryoprotectant. Cryoprotectants include DMSO,glycerol, polyethylene glycol, propylene glycol, glycerine,polyvinylpyrolidone, sorbitol, dextran, and trehalose. The exosomecompositions also retain potency after being frozen without usingspecial freezing protocols. Special freezing protocols include flashfreezing, programmable rate freezer, and freezing in an insulatedcontainer. The exosome compositions are frozen in buffer or culturemedia. Buffers include physiologically acceptable buffers such asphosphate buffer, histidine buffer, citrate buffer, acetate buffer, andother suitable buffers. In some cases exosome compositions disclosedherein are lyophilized.

Methods of isolation or purification of exosome compositions using CD39binding agents such as paracrine signaling exosome compositionsdisclosed herein include combining compositions comprising exosomes, forexample in vitro exosomes, and SDC2+ mesenchymal stem cells (e.g., SDC2+mesenchymal stromal stem cells). In some instances, methods of isolationor purification of exosome compositions include combining compositionscomprising in vitro exosomes and regulatory T cells. Regulatory T cellsinclude CD25+ regulatory T cells, CD4+ regulatory T cells, FoxP3+regulatory T cells, CD25+CD4+ FoxP3+ regulatory T cells, andcombinations thereof. In some instances, methods of isolation orpurification of exosome compositions include combining compositionscomprising in vitro exosomes, SDC2+ mesenchymal stem cells (e.g., SDC2+mesenchymal stromal stem cells), and regulatory T cells.

Disclosed herein are methods of isolation or purification ofcompositions comprising exosomes using CD39 binding agents such asparacrine signaling exosomes in a therapeutically effective amount Atherapeutically effective amount of exosomes, in some cases, ranges from10⁶-10⁸ exosomes, for example 10⁶, 10⁷, 10⁸, or more exosomes in thecomposition. In some cases, a therapeutically effective amount ofexosomes ranges from 1 mg to 700 mg of exosomes, for example 1 μg, 10μg, 20 μg, 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 500 μg, 750 μg, 1 mg,2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg,300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or more exosomes in thecomposition. Isolation or purification of exosome compositions, in somecases, comprises concentrating the exosome composition to be diluted bythe individual prior to administration. In some cases, isolation orpurification of exosome compositions comprises diluting the exosomecomposition making it ready to be administered by the individual. Insome cases, methods of isolation or purification of exosome compositionsresults in single use vials or syringes. In some cases, methods ofisolation or purification of exosome compositions results in multipledoses are present in a single container.

Isolation or purification of exosome compositions such as paracrinesignaling exosome compositions using CD39 binding agent, in some cases,is accomplished with the addition of antibodies to SDC2, which has theeffect of increasing the yield of exosomes and increasing the proportionof isolated exosomes that comprise SDC2. Methods of isolation orpurification comprise incubating a composition comprising exosomes withan anti-CD39 antibody and an anti-SDC2 antibody and retaining only theexosomes bound to the anti-CD39 and the anti-SDC2 antibody. Anti-SDC2antibodies suitable for methods herein have the property of bindingspecifically to SDC2. Incubation of a composition comprising exosomeswith the anti-SDC2 antibody is done in a buffer suitable for specificbinding of the anti-SDC2 antibody to SDC2 and at a temperature that issuitable for antibody binding and exosome stability. In some cases, theincubation is done at room temperature. In some cases, the incubation isdone at 4° C. The incubation buffer comprises at least one or a buffer,a detergent, and a salt.

Some compositions comprising exosomes such as paracrine signalingexosomes from which the exosome composition is isolated or purifiedusing CD39 binding agents, in some cases, comprises a cell culture. Acell culture includes but is not limited to SDC2+ cells, mesenchymalstem cells, SDC2+ mesenchymal stem cells, SDC2+ mesenchymal stromal stemcells, CD39+ cells, CD39+ mesenchymal stem cells, CD39+ mesenchymalstromal stem cells, CD39+/SDC2+ cells, and combinations thereof. In somecases, the cell culture is genetically modified to overexpress SDC2 bysuitable methods. Methods of isolation or purification of exosomecompositions using anti-SDC2 antibodies comprise a method of isolatingthe SDC2 antibody-exosome complex from the starting material. SDC2antibody-exosome complexes are purified by methods including but notlimited to fluorescence activated cell sorting (FACS),immunoprecipitation, column purification using protein A beads, columnpurification using protein G beads, column purification usingbiotinylated beads and a biotinylated secondary antibody, and magneticbead based separation methods. Exosome compositions are then eluted fromthe antibody using a buffered salt solution having stringency sufficientto elute the exosome composition from the antibody. Buffered saltsolutions are removed from the exosome composition using a desaltingcolumn or dilation procedure. The resulting isolated, purified exosomecomposition is then diluted in a physiologically acceptable buffer orexcipient and frozen or otherwise stored at a temperature where theexosome composition with retain potency and stability.

Isolation or purification of exosome compositions such as paracrinesignaling exosome compositions, in some cases, is accomplished with theuse of antibodies to CD39, which has the effect of increasing the yieldof exosomes and increasing the proportion of isolated exosomes thatcomprise SDC2. Methods of isolation or purification comprise incubatinga composition comprising exosomes with an anti-CD39 antibody andretaining only the exosomes bound to the anti-CD39 antibody. Anti-CD39antibodies have the property of binding specifically to CD39. Incubationof a composition comprising exosomes with the anti-CD39 antibody is donein a buffer that promotes specific binding of the anti-CD39 antibody toCD39 and at a temperature that is optimal for antibody binding andexosome stability. In some cases, the incubation is done at roomtemperature. In some cases, the incubation is done at 4° C. Theincubation buffer comprises at least one or a buffer, a detergent, and asalt.

The composition comprising exosomes such as paracrine signaling exosomesfrom which the exosome composition is isolated or purified usingantibodies to CD39, in some cases, comprises a cell culture. A cellculture includes but is not limited to SDC2+ cells, mesenchymal stemcells, SDC2+ mesenchymal stem cells, SDC2+ mesenchymal stromal stemcells, CD39+ cells, CD39+/SDC2+ cells, and combinations thereof. In somecases, the cell culture is genetically modified to overexpress CD39 bysuitable methods. Methods of isolation or purification of exosomecompositions using anti-CD39 antibodies comprise a method of isolatingthe CD39 antibody-exosome complex from the starting material. CD39antibody-exosome complexes are purified by methods including but notlimited to fluorescence activated cell sorting (FACS),immunoprecipitation, column purification using protein A beads, columnpurification using protein G beads, column purification usingbiotinylated beads and a biotinylated secondary antibody, and magneticbead based separation methods. Exosome compositions are then eluted fromthe antibody using a buffered salt solution having stringency sufficientto elute the exosome composition from the antibody. Buffered saltsolutions are removed from the exosome composition using a desaltingcolumn or dilation procedure. The resulting isolated, purified exosomecomposition is then diluted in a physiologically acceptable buffer orexcipient and frozen or otherwise stored at a temperature where theexosome composition with retain potency and stability.

Isolation or purification of exosome compositions such as paracrinesignaling exosome compositions, in some cases, is accomplished usingultracentrifugation methods, such as preparative ultracentrifugation.Methods of isolation or purification of exosome compositions, compriseobtaining a population of cells such as stromal cells, SDC2+ cells,mesenchymal stem cells, SDC2+ mesenchymal stem cells, SDC2+ mesenchymalstromal stem cells, CD39+ cells, CD39+/SDC2+ cells, and combinationsthereof. In some cases, the cells have been genetically altered tooverexpress SDC2 and/or CD39. The media or supernatant of the cellculture is isolated or purified from the cell culture. Then, the mediaor supernatant is mixed with an appropriate salt or buffer to enhancethe separation efficacy in ultracentrifugation. The resulting mixture isadded to an ultracentrifugation tube which allows the mixture to safelyendure high centrifugal forces of about 100,000×g (or 100,000 times theforce of gravity) for 1 to 24 hours. Exosome compositions are foundconcentrated together and removed from the tube. In some cases, theexosomes are removed as a resuspended pellet from the tube. In somecases, the exosomes are visualized in the resulting density gradient andremoved by needle aspiration, or other method. The resulting exosomecomposition is purified from the ultracentrifugation buffer and dilutedin a physiologically acceptable buffer or excipient and frozen orotherwise stored at a temperature where the exosome composition withretain potency and stability.

Isolation or purification of exosome compositions such as paracrinesignaling exosome compositions, is accomplished in some cases usingultrafiltration. Some ultrafiltration methods are suitable, such asmethods involving concentration columns that allow passage of aqueousbuffers but not high molecular weight substances, such as exosomes. Someexamples of ultrafiltration methods of isolation or purification methodcomprise obtaining a population of cells such as stromal cells, SDC2+cells, mesenchymal stem cells, SDC2+ mesenchymal stem cells, SDC2+mesenchymal stromal stem cells, CD39+ cells, CD39+/SDC2+ cells, orcombinations thereof. In some cases, the cells have been geneticallyaltered to overexpress SDC2 and/or CD39 or have been provided withexogenous SDC2 and/or CD39 or a vector encoding SDC2 and/or CD39. Themedia or supernatant of the cell culture is isolated or purified fromthe cell culture. Then the media or supernatant is concentrated, such asby a factor of 30, for example 150 ml of starting media or supernatantresulting in 10 ml of concentrated exosomes, using a Stirred Cell Model8200 with 100,000 kDa Biomax polyethersulfone or Ultracel regeneratedcellulose membranes using nitrogen gas at 10 psi. The concentratedexosomes are then transferred to a collection device such as an AmiconUltra-15 100,000 kDa device and centrifuged, for example in an AllegraX-15R centrifuge at 4,000×g at 4° C., to concentrate the exosomes byanother factor of 20, for example 10 ml of concentrated exosomesresulting in a further concentrated 0.5 ml. Alternative columns andcentrifuges are substituted in appropriate cases.

In some cases, isolation or purification of exosomes compositions suchas paracrine signaling exosome compositions is accomplished usingautomated systems of manufacturing. In some cases, automatedmanufacturing is comprises using the Terumo Quantum Cell ExpansionSystem.

Stromal Stem Cell Compositions

Provided herein are compositions, such as compositions comprising SDC2+stromal stem cells, bound to a CD39 binding agent. Some suchcompositions comprise SDC2+CD39+ stromal stem cells or CD39+ stromalstem cells. In some cases, at least 20% of the stromal stem cells in thecomposition are SDC2+. In some cases, at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 99%, or more of the stromal stem cells are SDC2+. Insome cases, at least 20% of the stromal stem cells in the compositionare CD39+. In some cases, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, or more of the stromal stem cells are CD39+. In somecases, at least 20% of the stromal stem cells in the composition areSDC2+CD39+. In some cases, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, or more of the stromal stem cells are SDC2+CD39+. Theproportion of stromal stem cells in the composition comprising SDC2 isdetermined by immunofluorescence, for example flow cytometry, magneticactivated cell sorting, fluorescence microscopy, or other suitablemethod.

CD39 binding agents herein, in some cases, comprise antibodies, such asanti-CD39 antibodies or fragments thereof. In some cases, antibodies areraised against a CD39 antigen. In some cases, antibodies comprise avariable domain that specifically binds to at least one mammalian CD39protein. In some cases, antibodies specifically bind to at least one ofa human, a mouse, a rat, or an equine CD39 protein. In some cases, theantibody is conjugated to a fluorophore. In some cases, the antibody isconjugated to a bead.

SDC2 refers to a gene encoding the syndecan-2 protein (also frequentlyreferred to herein and elsewhere in the art as SDC2). Syndecan-2, or‘the SDC2 protein’ or simply SDC2, is a transmembrane type I heparinsulfate proteoglycan. Additional synonyms for syndecan-2, aside from‘the SDC2 protein’ or SDC2, include HSPG, CD362, HSPG1, and SYND2.Generally, as used herein SDC2 refers to the protein or a recognizablefragment thereof unless otherwise indicated, for example by reciting‘the SDC2 gene,’ ‘the SDC2 transcript,’ ‘an SDC2 antibody.’Additionally, SDC2 is identified by its polypeptide sequence in thesequence listing that accompanies this specification.

Stromal cell compositions herein, in some cases, are modified toincrease the therapeutic efficacy of the stromal stem cell composition.In some cases, cells are genetically modified to overexpress an apyrasesuch as CD39 or an alternative apyrase as discussed herein. In somecases, cells are genetically modified to overexpress CD39L3. Geneticmodification of stromal cells is accomplished by methods including butnot limited to transfection of the stromal cells with one or moreplasmids that comprise the CD39 or CD39L3 coding sequence and apromoter, such as a CMV, SV40, EF1a, or CAG promoter. Geneticmodification of stromal cells, in some cases, is accomplished byinfection of the stromal cells with a virus that comprises the CD39 orCD39L3 coding sequence and a promoter.

SDC2 has three domains: an extracellular domain at amino acids 19-144, atransmembrane domain at amino acids 145-169, and a cytoplasmic domain atamino acids 170-201. SDC2 has been implicated in the mediation of cellbinding, cell signaling, and cytoskeletal organization. SDC2 has beendemonstrated to be necessary for internalization of HIV-1 TAT protein.

Stromal stem cell compositions disclosed herein, in some cases, comprisea cryoprotectant or cryopreservative. Cryoprotectants include DMSO,glycerol, polyethylene glycol, propylene glycol, glycerine,polyvinylpyrolidone, sorbitol, dextran, trehalose, and commercialformulations such as CryoStor from Biolife solutions. Stromal stem cellcompositions herein retain potency after being frozen using specialfreezing protocols. Special freezing protocols include flash freezing,programmable rate freezer, and freezing in an insulated container. Thestromal stem cell compositions are in some cases frozen in buffer orculture media having an added cryoprotectant. Buffers includephysiologically acceptable buffers such as phosphate buffer, histidinebuffer, citrate buffer, acetate buffer, Hypothermasol from BiolifeSolutions and other suitable.

Stromal stem cell compositions disclosed herein are formulated in aphysiologically acceptable buffer and in some cases supplemented by atleast one excipient. Non-limiting examples of excipients includesucrose, trehalose, polyethylene glycol, a polysaccharide, a carrierprotein, an inert protein, dextran, hydroxyl ethyl starch (HETA),PEG-4000, gelatin, PLGA, Eudragit RS 100 Nanoparticles, and combinationsthereof. Such stromal stem cell compositions are stored at a temperaturedetermined to be most stable (i.e., wherein the stromal stem cellcomposition retains highest potency, or retains potency for the longestperiod of time, or otherwise optimizes a desired trait). In some cases,addition of at least one excipient allows the composition to retainpotency, such as paracrine signaling potency, when stored at a highertemperature than otherwise would be possible.

Some stromal stem cell compositions such as CD39+, SDC2+, or SDC2+CD39+stromal stem cell compositions isolated using apyrase extracellularbinding domain agents such as CD39 binding agents disclosed herein, insome cases, comprise SDC2+ stromal stem cells and another mammaliancell. The other mammalian cell combined SDC2+ stromal stem cells are insome cases regulatory T cells, such as CD25+ regulatory T cells, CD4+regulatory T cells, FoxP3+ regulatory T cells, CD25+CD4+ FoxP3+regulatory T cells, or combinations thereof.

Compositions comprising a wide range of CD39+, SDC2+, or SDC2+CD39+stromal stem cells are disclosed herein. Some compositions compriseSDC2+ stromal stem cells in a therapeutically effective amount. In somecompositions, the amount of SDC2+ stromal stem cells ranges from10{circumflex over ( )}3-10{circumflex over ( )}8 SDC2+ stromal stemcells, for example 10{circumflex over ( )}3, 10{circumflex over ( )}4,10-5, 10{circumflex over ( )}6, 10{circumflex over ( )}7, or10{circumflex over ( )}8 SDC2+ stromal stem cells, or more SDC2+ stromalstem cells in the composition. SDC2+ stromal stem cell compositions, insome cases, are concentrated to be diluted by the individual or thehealth care provider prior to administration. In some cases, SDC2+stromal stem cell compositions are diluted and ready to be administeredby the individual or health care provider. In some cases, SDC2+ stromalstem cell compositions are contained in single use vials, syringes, orIV bags. In some cases, multiple doses are present in a singlecontainer.

A therapeutically active CD39+, SDC2+, or SDC2+CD39+ stromal stem cellcomposition disclosed herein, in some cases, comprises animmunosuppressive drug. Immunosuppressive drugs contemplated hereininclude but are not limited to a glucocorticoid, a cytostatic, anantibody, an immunophilin inhibitor, cyclosporin, tacrolimus, sirolimusand interferon, an opioid, a TNF binding protein, a cyclooxygenaseinhibitor, an antihistamine, an antimetabolite, folic acid,methotrexate, a purine analogue, a pyrimidine analogue, a proteinsynthesis inhibitor, mycophenolate, a cytotoxic antibiotic, a steroid,an anti-TNF antibody, a TNF inhibitor, and an NSAID. Some SDC2+ stromalstem cell compositions comprise anti-TNF antibodies, including but notlimited to infliximab, adalimumab, certolizumab, and golimumab. SDC2+stromal stem cell compositions comprise TNF inhibitors including but notlimited to etanercept, xanthine derivatives, and bupropion. In somecases, SDC2+ stromal stem cell compositions comprise at least one NSAID.In some cases, SDC2+ stromal stem cell compositions comprise at leastone steroid.

Exosome Compositions

Provided herein are compositions, such as therapeutically active,compositions comprising exosomes, for example in vitro exosomes, such asSDC2+ exosomes, bound to a CD39 binding agent. In some cases theexosomes comprise components that mediate, effect or inhibit paracrinesignaling. In some cases, at least 20% of the exosomes in thecomposition are SDC2+ or comprise SDC2. In some cases at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of the exosomes compriseSDC2. In some cases at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, or more of the exosomes comprise CD39. SDC2, in some instancesis found on the surface of the exosome. In some instances, SDC2 is foundat the interior of the exosome. The proportion of exosomes in thecomposition comprising SDC2 is determined by immunofluorescence, forexample flow cytometry, electron microscopy, or other suitable method.

CD39 binding agents herein, in some cases, comprise antibodies, such asanti-CD39 antibodies. In some cases, antibodies are raised against aCD39 antigen. In some cases, antibodies comprise a variable domain thatspecifically binds to at least one mammalian CD39 protein. In somecases, antibodies specifically bind to at least one of a human, a mouse,a rat, or an equine CD39 protein. In some cases, the antibody isconjugated to a fluorophore. In some cases, the antibody is conjugatedto a bead.

SDC2 refers to a gene encoding the syndecan-2 protein (also frequentlyreferred to herein and elsewhere in the art as SDC2). Syndecan-2, or‘the SDC2 protein’ or simply SDC2, is a transmembrane type I heparinsulfate proteoglycan. Additional synonyms for syndecan-2, aside from‘the SDC2 protein’ or SDC2, include HSPG, CD362, HSPG1, and SYND2.Generally, as used herein SDC2 refers to the protein or a recognizablefragment thereof unless otherwise indicated, for example by reciting‘the SDC2 gene,’ ‘the SDC2 transcript,’ ‘an SDC2 antibody.’Additionally, SDC2 is identified by its polypeptide sequence in thesequence listing that accompanies this specification.

SDC2 has three domains: an extracellular domain at amino acids 19-144, atransmembrane domain at amino acids 145-169, and a cytoplasmic domain atamino acids 170-201. SDC2 has been implicated in the mediation of cellbinding, cell signaling, and cytoskeletal organization. SDC2 has beendemonstrated to be necessary for internalization of HIV-1 TAT protein.

While exosome compositions described herein, in some cases, are derivedfrom cells, the exosome compositions do not necessarily comprise livingcells. Cell-free exosome compositions, therefore, are non-tumorigenic,that is, they do not increase the susceptibility of a subject todeveloping a tumor or cancer, because they do not comprise cells capableof differentiating into tumor cells. In alternative compositions, theexosomes are supplemented with cells, such as mesenchymal stromal cells,that contribute to anti-inflammatory activity or paracrine signalingactivity of the compositions.

Exosome compositions disclosed herein retain potency or activity, suchas paracrine signaling activity, after being frozen or cryopreserved,often without the use of a cryoprotectant. Cryoprotectants include DMSO,glycerol, polyethylene glycol, propylene glycol, glycerine,polyvinylpyrolidone, sorbitol, dextran, trehalose, and commercialformulations such as CryoStor from Biolife solutions. The exosomecompositions also retain potency after being frozen without usingspecial freezing protocols. Special freezing protocols include flashfreezing, programmable rate freezer, and freezing in an insulatedcontainer. A benefit of the durability of the exosome compositions isthat they are more easily frozen and are frozen without cryoprotectants,resulting in compositions that are more durable, more easily and cheaplymade, and less likely to suffer from batch variation resulting from lossof activity due to a defect in freezing protocol or composition. Theexosome compositions are in some cases frozen in buffer or culturemedia. Buffers include physiologically acceptable buffers such asphosphate buffer, histidine buffer, citrate buffer, acetate buffer,Hypothermasol from Biolife Solutions and other suitable buffers. In somecases exosome compositions disclosed herein are lyophilized.

Exosome compositions disclosed herein are formulated in aphysiologically acceptable buffer and in some cases supplemented by atleast one excipient. Non-limiting examples of excipients includesucrose, trehalose, polyethylene glycol, a polysaccharide, a carrierprotein, an inert protein, dextran, hydroxyl ethyl starch (HETA),PEG-4000, gelatin, PLGA, Eudragit RS 100 Nanoparticles, and combinationsthereof. Such exosome compositions are stored at a temperaturedetermined to be most stable (i.e., wherein the exosome compositionretains highest potency, or retains potency for the longest period oftime, or otherwise optimizes a desired trait). In some cases, additionof at least one excipient allows the composition to retain potency, suchas paracrine signaling potency, when stored at a higher temperature thanotherwise would be possible.

Some exosome compositions such as paracrine signaling exosomecompositions disclosed herein, in some cases, comprise in vitro exosomesand SDC2+ mesenchymal stem cells (e.g., mesenchymal stromal stem cellsisolated based upon expression of CD39). The cells combined to in vitroexosomes are in some cases regulatory T cells, such as CD25+ regulatoryT cells, CD4+ regulatory T cells, FoxP3+ regulatory T cells, CD25+CD4+FoxP3+ regulatory T cells, or combinations thereof. In some instances,exosome compositions comprise in vitro exosomes, SDC2+ mesenchymal stemcells (e.g., SDC2+ mesenchymal stromal stem cells isolated based uponexpression of CD39), and regulatory T cells (e.g., regulatory T cellsisolated based upon expression of CD39).

Compositions comprising a wide range of exosomes are disclosed herein.Some compositions comprise exosomes in a therapeutically effectiveamount. In some compositions, the amount of exosomes ranges from 10⁶-10⁸exosomes, for example 10⁶, 10⁷, 10⁸, or more exosomes in thecomposition. In some cases, a therapeutically effective amount ofexosomes ranges from 1 mg to 700 mg of exosomes, for example 1 mg, 10mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 750 mg, 1 mg,2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg,300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or more exosomes in thecomposition. Exosome compositions, in some cases, are concentrated to bediluted by the individual prior to administration. In some cases,exosome compositions are diluted and ready to be administered by theindividual. In some cases, exosome compositions are contained in singleuse vials or syringes. In some cases, multiple doses are present in asingle container.

Some exosome compositions disclosed herein comprise additional proteins,such as proteins that contribute to therapeutic efficacy, or thatmediate paracrine signaling to effect therapeutic efficacy. Proteinsinclude but in some cases are not limited to IL-12, suppressor ofcytokine signaling (SOCS), p53, PTEN, CD52, TSC1, FOXP3, Soluble ImmuneResponse Suppressor (SIRS), TGFB, CD39L3, CD73, and Maspin. In somecases, exosomes comprise at least one of protein selected from the listconsisting of UBA6, ESYT2, SSC5D, STMN1, STMN2, PRNP, VEGFA, ADD1, NBL1,MINOS1-NBL1, XIRP2, VPS37C, MARS, BST1, MAP1LC3B, MAP1LC3B2, RPSA,RPSAP58, BPNT1, ABI1, SEPT8, NUDT5, WBP2, SPTAN1, ATP1B1, DYNLT3, YIF1A,SEC61A2, SEC61A1, DOCK11, NDE1, NDEL1, UFD1L, SNX1, KIAA1217, CNIH4,CRYZL1, PRAMEF26, PRAMEF11, PRAMEF6, PRAMEF5, PRAMEF23, PRAMEF9,PRAMEF4, ANXA6, CD9, AMOT, PPP2R4, SELENBP1, PSMD4, PIP5K1A, PIPSL,MLLT4, GSK3A, RTN1, MPP1, DSCR3, SUMO2, SUMO3, SUMO4, PPP5C, AIMP2,TUBA4A, RPL23A, CASP3, FAM171A2, KIAA1324L, RAB34, C2orf74, DRG2, MBP,PTTG1IP, MBOAT7, CSNK1E, CSNK1D, EIF3L, EIF3D, UBE2I, CPNE1, APOC3,ARCN1, XRCC6, PSMD10, CROCC, NRD1, TSPAN2, MTOR, DYNLRB1, DYNLRB2,PEA15, POSTN, ARHGEF7, LPHN2, SEPT6, CD58, LGALS8, CD55, C1orf123,EPS15, MUC1, PSAP, GME, TXNRD1, HMGB1, HMGB1P1, RUVBL2, SLC29A1, EIF3S3,EIF3H, EIF3F, METRNL, CA12, PLTP, FNTA, SNRPN, SNRPB, HARS, AP1G1,CDIPT, CNN2, LARS, EIF2A, NAPG, CNN3, IDH2, ULK3, RPS6KA3, NPLOC4, CANX,EIF2B1, PIP4K2A, MYRF, TMEM165, EPN3, TARDBP, RAB5A, SNRPD3, SNX6, CS,LPAR1, AKR1C1, AKR1C2, AKR1C3, SACM1L, CARS, CHURC1-FNTB, FNTB, PCBP2,PCBP3, STAT2, NQO1, MAT2A, STRAP, IL6, SERPINB1, ABHD14A, ABHD14A-ACY1,ACY1, PTGES3, PGD, NPC2, HIST1H2BN, HIST1H2BL, HIST1H2BM, HIST1H2BH,HIST2H2BF, HIST1H2BC, HIST1H2BD, H2BFS, HIST1H2BK, HIST2H2BE, HIST1H2BB,HIST1H2B0, HIST1H2BJ, STK24, PLOD1, ELMO2, ZDHHC20, FAM98A, ANXA7,SLC2A5, PLSCR1, RASA1, DKFZp434N071, SLC9A1, MTAP, TMBIM1, SERINC3,AHSA1, QARS, ARL1, DNAJB1, NMT2, NMT1, FXR1, HNRNPC, RALYL, HNRNPCL1,TGFBR1, ME1, COPB2, TKT, RALB, DBF4B, LRCH3, PNPO, RBM4B, MST4,SERPING1, GALK1, PBXIP1, AQP1, SRSF3, FARSA, EML4, PPP1R7, STEAP2,GUCD1, PDIA6, SIRT2, QPCT, TSPAN9, RAN, EIF3C, EIF3CL, SEPT10, CAP2,NTM, HBS1L, RCN1, ATP6V1A, RNF14, SLC26A4, PTPRA, ATP6V0A1, MFSD8,TOM1L2, SGCE, CYTH3, TSPAN5, EXOC4, PPP6C, ALAD, PFKM, ISYNA1, PCYOX1,ATP6AP2, CAST, RPN1, INPP5K, SLC6A9, LPXN, AKT1, RRAS2, DECR1, SH3KBP1,NUBP2, PMM2, SCFD1, ACP2, PITPNB, GYS1, USP7, GPRC5B, RAB1A, EMB, EBF2,PCMT1, NAP1L1, SH3PXD2A, CCT4, GALK2, DLST, SH3GLB2, SCARB1, CCDC122,HSPE1, PPIL3, PTMA, TAX1BP1, EVA1A, FAM126A, TCEB2, IGLL5, DNPEP,DIAPH1, DISC1, TSNAX, DCUN1D1, PFN2, SRI, CNTLN, EEF1E1, EEF1E1-BLOC1S5,PTPN12, EIF4G1, TMEM248, TPST1, CPA4, MID1, CXCL8, RPL37A, KIAA0319L,IGF1R, TMEM98, PFN2, TNPO3, ATP6V1E1, RARRES2, ITGB6, APPL1, IFT57,TFPI, PSPH, QPRT, MEST, LTBP1, PRPSAP2, MTMR2, GPS1, CYCS, ITM2C, TYMP,APEH, OXSR1, PPM1B, TFG, ARVCF, STARD3NL, KIAA0195, MTPN, DGKA, MASP1,FARP1, FAM3C, DDX17, RPL24, UBA5, SEC14L2, SEC14L3, TIA1, TIAL1, BTN3A3,BTN3A2, BTN3A1, CD63, LEPROTL1, TENC1, ARMC9, EPHA5, EPHA3, TMEM106B,RPL35A, TMEM50B, ALB, EIF4G2, GNPDA1, GNPDA2, CAMK2D, CAMK2B, CAMK2A,GPM6A, ABCE1, CLDND1, MFSD10, RPL9, NECAP2, CTBP1, CTBP2, SPON2, SNF8,DCTD, RELL1, LMAN2, EIF4E, TTC37, IGJ, ALG13, RPS23, SRP72, CALCOCO2,PAIP1, RNASET2, SEPT11, SEC31A, MCC, CXCL6, CXCL5, HAPLN1, CD14,COL12A1, CLTB, ELOVL5, EIF3E, LYPLA1, PFDN1, TCEB1, SORBS3, ERLIN2,ERLIN1, ENY2, RPL30, PLAA, FABP4, TBCA, MAT2B, SKP1, COPSE, SQSTM1,AP3D1, BLMH, RAI14, MAP4K4, FES, FER, SEC24C, ABI2, RPL14, CD44, SEPTI,PTPRM, GLB1, SLC43A3, EIF4A2, PABPC1, PABPC4, PAPSS2, ATP2C1, TNS1,TNS3, THBS4, HEPH, PSEN1, XPO7, PLAU, ITGA2, STX3, PPP3CA, RPS24, PLOD2,MARK2, MARK1, MARK3, GPX8, BZW2, GDI2, CSNK2A1, CSNK2A3, DKK3, CDK14,CDK4, CDK3, CDK1, CDK16, CDK12, CDK15, CDK9, CDK18, CDK13, SF3A3, ASB2,CAPN5, CYFIP2, KLC1, MYO6, IQGAP2, ADAM23, HYI, TRIO, MGLL, DCTN1,NIF3L1, PI4K2A, NACA, GPR84, MGRN1, PACS2, RBBP7, RBBP4, NLN, COL6A3,HNRNPH1, MDH2, PTPRD, PTPRS, MYO1B, PHLDB2, SRP9, ATP11A, PPIE, DIP2A,EPB41, DTNB, TNS1, RND3, PPP2R5D, MANBA, AP2M1, APP, AAK1,C1QTNF3-AMACR, C1QTNF3, TSN, KIDINS220, DPM1, GSTM2, PLSCR4, EPB41L2,PRKCDBP, MUC15, PDE8A, THY1, TCP11L1, RPL27A, CRYAB, AAMDC, TMEM126B,EEF1D, SCYL1, PPP6R3, PRMT1, DCAF5, NUCB2, TSTA3, RPL8, HYOU1, RAB1B,NPEPPS, MDK, VKORC1, AASDHPPT, RNF141, TYK2, USP47, WLS, PSMC3, TSPAN4,STT3A, CD59, LRP8, RAE1, MVB12A, IFITM2, IFITM3, IFITM1, MAPK3, PFDN4,IFT46, EFEMP2, NSFL1C, FRYL, ARRDC1, PITPNA, CCT2, ADA, PCDH7, KRT17,SMAD5, TMED2, MPI, ITFG1, METAP1, RPTOR, HN1, GALNT1, COPS7A, KPNA6,KPNA5, KPNA1, OTUB1, ATP6V0D1, PXN, MACF1, SLC3A2, PPP3CB, GLTP, FERMT3,FBLN2, SEMA3C, CALD1, DCTN2, UACA, TENM2, MTHFD1, CBS, EIF3A, HMBS,SEC23A, PPP2R1A, TSG101, AP3S1, TMX3, VPS26A, VPS37B, SUGT1, SLC8A1,STK4, LSAMP, CDC42BPA, B2M, ATL3, TBC1D9B, FARSB, CDK17, VDAC3, CYB561,MFGE8, FZD6, BCAR1, TNIK, RPS10, RPS10-NUDT3, ST13, ST13P4, ST13P5,RBM38, PIP4K2C, CAD, PRKAG1, TMBIM6, DDX39B, DDX39A, DDX39, hCG 2005638,C12orf75, OCC1, C12orf10, CSRP2, COPZ1, SCYL2, PLXNA1, IGFL2, PTPRB,CHMP1A, RPL18, SLC25A3, SLC38A1, VPS29, PPP1CC, KIAA1033, KRT18, CTDSP2,FMNL3, PDE6H, MYL6, HNRNPA1, MYH10, RASA3, SDK1, BRE, GOLIM4, RANGAP1,RTN4, IGF2BP2, EX005, ABHD14B, PRKD2, PRKD1, PRKD3, TM4SF1, RNF149,ARPC4, ARPC4-TTLL3, CMTM7, DTNA, DTNB, PAM, TRAPPC2P1, TRAPPC2, ATP6V1H,DPP3, RPL21, TJP1, HBA2, GOLT1B, BAGS, PSMA6, UBE2V1, UBE2V2, MPP5,GNG2, FBN3, ACYP1, PTGER2, VPS33B, LTBP2, SRP54, GMFB, FRMD6, FBLN5,GNPNAT1, SHMT2, SLC7A7, SULF2, LAMB1, COMP, SBF1, TTC7A, PDIA3, COBLL1,XPNPEP1, DNM1L, GRK5, GRK6, GRK4, CDSN, MVB12B, ALDH1A3, HP, HPR, AMPD2,KCNMA1, FN1, IMPDH2, APMAP, CC2D1B, TBC1D8B, COL12A1, PTPRF, CALM2,CALM1, CALM3, HNRNPD, ATP10D, FST, COL14A1, VEGFA, CTSC, CTSF, MYCT1,CD40, RPS2, SCP2, CRIP2, C1R, REXO2, M6PR, LOC388849, IFT81, DHRS7,PPP2R5C, HECTD1, ETFA, WDR61, GMPR2, LDLR, RPS27L, RPS27, PSMA4, ANP32A,ANP32B, SPPL2A, PSME1, COMMD4, SPNS1, SLC9A3R2, RPS15A, CARHSP1, FUS,TAF15, HAGH, HNRNPUL2-BSCL2, HNRNPUL2, GSPT1, UBFD1, LRRC57, DDX19A,DDX19B, HAPLN3, HAPLN4, BOLA2B, BOLA2, STXBP5, GCA, CHIA, ABCB6, COL5A1,WDR44, PDIA3, ZPR1, YIPF4, AP1S1, AP1S2, MMS19, CTDSP1, TSPAN15, MANF,POFUT2, PLOD3, MITD1, STRADB, PRDX4, SRPRB, MAGI1, ATP11B, MFSD1, IAH1,EFR3A, BRCC3, TXNDC17, NSF, PCYT2, CRK, MLKL, TFAP4, TOM1L1, EIF5A,EIF5AL1, EIF5A2, SRR, RPS15A, CCDC43, BAIAP2, SLC12A4, RPS13, MATN2,PI4KA, KIAA0368, ECM29, PSMD9, KIF5A, KIF5C, GPR176, PARVA, ITGA7, FHL2,MY018A, LEKR1, GAS6, OLA1, TGOLN2, SYT1, STRA6, PSD3, PPP2R5E, IARS,NT5C, COPZ2, TANC2, RPL17, SRSF1, RPL38, FBXL20, RPL19, ARHGDIA, AKT2,SNRPD1, VWA1, ACE, ERBB2, CDKN2A, CDKN2B, MYL12A, MYL12B, YES1, RPL13,FNBP1L, TMEM8A, CUL4B, MYO1D, KATNAL2, SMAD4, RPS15, RPL22, TBCB, PKN1,SEPT9, SYNGR2, PRKCSH, RAD23A, EIF1, EIF1B, ACTG1, MRI1, PIN1, STAT3,UBXN6, DAZAP1, PDCD5, CARM1, CDC37, GPX4, PSENEN, RAB27B, COPE, ARHGEF1,NUMBL, HNRNPM, AP2S1, EMP3, RCN3, GGCT, JOSD2, CLEC11A, RPS5, MYO9B,AXL, PLAUR, RPL18A, SPATA22, EPS15L1, CHMP2A, KDELR1, KDELR2, RPS16,DPP9, TBC1D17, PAFAH1B3, ACOT7, MYO1C, SNAP23, STXBP3, AP3B1, TNFRSF10A,PSMD11, PSMD12, PGRMC1, CLIC1, QSOX1, IP05, RTCA, AGRN, PSMD14, KPNA3,STK25, KRIT1, SDCBP, SDCBP, DDX3X, DDX3Y, CYR61, KPNA4, PDXK, CLDN4,CLDN9, CLDN6, CLDN3, PPAP2A, PPAP2B, ISLR, TXNDC9, HSPB6, ADAM10,ITGB1BP1, PRMT5, SLC9A3R1, TNFRSF10B, NRP1, MRAS, PSMA7, SCAMP3,TAX1BP3, GIPC1, CASK, HGS, PPP1R12A, PPP1R12B, XPO1, PLXNB2, NPC1,SCAMP1, SCAMP2, ARPC1B, ARPC2, ARPC3, PGRMC2, PFDN6, LAMA5, LEPROT,RER1, SURF4, INPPL1, NCAM2, STX7, SLC16A3, SLC31A1, ABCC3, ABCC4, P4HA2,YKT6, ARPC5, FLRT2, PLXNB1, PHGDH, ADAM12, GPR39, DYNC1LI2, PSMD3,PAPSS1, B4GALT5, TGFB1I1, TXNL1, TPD52L2, FIBP, AKR7A2, EPB41L2, ATP8B1,ATP8B4, DENR, XPOT, TSPAN6, ASNA1, ACTN4, KDELR3, SGTA, NARS, LANCL1,CALU, EDIL3, AHCYL1, AHCYL2, SPAG9, MAPK8IP3, PIP5K1C, DFNA5, NRP2,ACSL4, SNX3, ADCY9, SYNCRIP, HNRNPR, GREM1, EXOC3, PLIN3, SLC16A7, UGDH,CTNND1, SNX2, USO1, TOM1, PRAF2, EIF5B, DNAJA2, CUTA, SRGAP2, SRGAP2C,PLXNA2, WDR1, FZD7, SLIT3, ROCK2, CPNE3, DNAJC13, USP12, SEMA7A, PDCD6,ATP6V1G1, ATP6V1G2-DDX39B, ATP6V1G2, VPS4B, SH3BGRL, FLNB, SEC22B,ERLIN1, GPC4, CLDN11, TIPRL, RP2, SLC22A3, EIF3J, CBR3, IDH1, ATRN,STAM2, ARL6IP5, DCTN3, FLOT1, CPD, GLRX3, STC2, CIAO1, DDAH1, STK10,GFPT2, SLIT2, SEC24D, FARP2, DKK1, ABCA8, ENDOD1, AP2A2, PRSS23, S1PR2,UBL3, VAMPS, RTN3, VAPB, MPZL1, PGLS, ATG7, LYPLA2, IP07, PGM3, APOM,FMNL1, ABCA1, SEC24A, SFT2D2, ACSL3, STAMBP, AP2A1, TMEM50A, BAG2, BAG3,CLDN1, CLIC3, TSPAN13, TSPAN31, DDAH2, ITGBL1, RECK, LDHA, ALDH1A1,GLUD1, GLUD2, CYB5R3, GSR, SOD1, F13A1, PNP, HPRT1, GOT2, EGFR, PGK1,AK1, C1R, F10, PLAT, ASS1, C3, TIMP1, CST3, CSTA, NRAS, HRAS, KRAS,TGFB1, PENK, NPY, IGF2, IL1B, IGHG1, IGHG3, COL1A1, COL3A1, COL4A1,LMNA, APOE, SLC4A1, FN1, FN1, FN1, RBP4, ORM2, ORM1, TFRC, FTL, FTH1,MT1X, MT1G, MT2A, MT1M, MT1E, MT1H, MT1A, ANG, VTN, CAT, ALDOA, CSTB,ANXA1, APOB, SOD2, OAT, KRT1, GAPDH, ASL, CAPNS1, HSPB1, RPN2, GNAI2,ATP1A1, ARG1, ITGB3, S100A8, SERPINB2, SERPINE1, ISG15, ALPL, EIF2S1,ICAM1, RPLP1, RPLP2, RPLP0, RPLP0P6, FABP3, ITGB1, PRKCB, MYL1, MYL3,COL5A2, UROD, INSR, FYN, GSN, GSN, S100A9, S100A6, ENO1, PYGL, GPI,NPM1, TPM3, ITGAV, LPL, SERPINE2, SERPINE2, EPHX1, DBI, LDHB, GPX1,P4HB, CTSD, ANXA2, ANXA2P2, CAPN1, TUBB, DCN, PFN1, BPGM, APRT, EPRS,CTSB, HSP90AA1, LYN, THBS1, HSPA1A, COL1A2, ANXA6, RHOC, PFKM, HSP90AB1,ASNS, MMP2, SOD3, MME, INHBA, MGP, ITGA2B, COL4A2, MFI2, ITGA5, VIM,RPS17L, RPS17, GNAI3, ANXA5, FGF2, ENO2, GSTP1, SNRPC, CXCL1, LGALS1,RBP1, SPARC, GSTM1, GSTM4, TPM1, TPM1, CLTA, ANXA4, CNP, PDGFRB, CIS,UCHL1, LTA4H, ALDOC, HIST1H2AJ, HIST1H2AH, H2AFJ, HIST2H2AC, HIST2H2AA3,HIST1H2AD, HIST1H2AG, HIST1H2AC, HIST3H2A, HIST1H2AB, RAP2A, SRGN,TROVE2, RRAS, BCL2, TXN, CTSA, PRKAR1A, ESD, HSPD1, CLU, HAPLN1, HSPA5,LAMC1, HSPA8, SLC2A1, SLC2A3, SLC2A14, LIMPS, PYGB, RALA, SPTB, LAMP1,G6PD, DMD, IGF2R, ADH5, PRPS2, PCNA, COL11A1, COL6A1, COL6A2, COL6A3,PIP, ANXA3, ACTN1, SRC, PEPD, GP1BB, LAMP2, RNH1, BMP1, NCAM1, VCAN,VCAN, VCAN, ITGA4, EEF2, PDIA4, P4HA1, TPT1, F3, PLS3, PRKAR2A, MIF,CD99, HGF, FDPS, CPM, NID1, AKR1A1, PKM, PKM, PKM2, HSP90B1, IDE, DARS,JUP, AKR1B1, ANPEP, PVR, RAC2, MYOD1, B4GALT1, EZR, UCHL3, CD46, CD46,NME1, VEGFA, DSP, TIMP2, CBR1, PDGFRA, ATP2A2, FAH, HSPA6, RHOQ, GOT1,PRKCA, ITGA2, GJA1, PRKACB, PRKACA, KIN27, PRKACG, CAPN2, GAP43, CTPS1,ENG, PFKL, GM2A, LGALS3, IGFBP3, FLT1, TCP1, IGFBP2, ITGB5, ARF4, RPL7,VCL, PGAM1, SDC1, CDH2, GNAZ, VCAM1, NCL, GGT1, GGT3P, GGT2, SRM,CSNK2A2, ATP2B1, EIF2S2, RAB3A, RAB3B, RAB4A, RAB6A, RAB6B, NPR2, PSMB1,COL5A1, PTMS, GSTM3, ATP6V1B2, ATP6V1C1, CSRP1, FLNA, ACO1, IRP1, S1PR1,TNFAIP3, NT5E, TBXA2R, VDAC1, BGN, COMT, TGM2, OSBP, GART, PAICS, UBA1,NME1-NME2, NME2, NME1, ENPP1, HNRNPA2B1, IGFBP4, FBLN1, ITGA6, PPIB,WARS, RPS3, JAK1, PTPRG, AHCY, CFL1, ATP2B4, LAMA2, EEF1B2, IGFBP5,ACP1, TNC, MYL9, F2R, AZGP1, RPS12, FAS, DNAJB2, PSMA1, PSMA2, PSMA3,ITGA3, PTX3, MSN, DDX6, CTNNA2, S100A4, MGAT1, PTBP1, TARS, VARS, EEF1G,STOM, YWHAQ, MARK3, ATP6VOC, DPP4, RPL10, COL8A1, CD82, CALR, PSMB8,PSMA5, PSMB4, PSMB6, PSMB5, LOX, MAPK1, LAP3, TPP2, IMPA1, CTGF, EPHA2,EPHB2, SHC1, SHC2, CRABP2, MARCKS, GNA11, PRDX6, BLVRB, PRDX5, DDT,DDTL, PRDX3, RPL12, ECHS1, CMPK1, PEBP1, BDKRB2, HLA-A, HLA-A, HLA-B,HLA-C, HLA-C, ADSS, LRPAP1, ADSL, SLC7A1, LCN1, LCN1P1, CORO1A, GDI1,S100A7, SDC4, DNAJA1, ATIC, CASP14, YWHAB, YWHAB, SFN, STIP1, S100A11,PRDX2, CCKAR, GBP1, STX2, KIF5B, ABCC1, RNASE4, SDC2, CD68, HSPA4, GPC1,CTNNA1, CTNNB1, SERPINB6, NF2, RDX, SPR, PTGS2, THBS2, KRT9, FBN1, MYH9,BSG, BSG, TIMP3, GLRX, KRT2, PSMC2, SLC16A2, CHI3L1, GGT5, ARL2-SNX15,ARL2, ARL3, MAP2K2, MAP2K1, RPL4, PGM1, GNL1, SERPINF1, TGFBR2, HPCAL1,HPCA, TAGLN2, TALDO1, HSPA9, RPS19, RPL3, COL15A1, COL18A1, CAPG, IL6ST,CCT6A, NNMT, MDH1, EIF2S3, EIF2S3L, CD200, WNT5A, CSK, GARS, STAT1,ECE1, SLC1A3, SLC1A1, SLC1A4, PAFAH1B1, PTGIR, MCAM, RANBP1, NAMPT,NAMPTL, PSMC4, PPIC, VDAC2, USPS, MAPK9, MAPK10, CRKL, GSTM5, BDKRB1,RPL5, RPS9, MAP1B, NEDD4, UTRN, IQGAP1, RABIF, CAPZA2, CAPZB, EIF1AX,EIF1AY, RPL29, XDH, SLC6A8, LIMS1, GLIPR1, CXCL12, PREP, TFPI2, GCLM,CD151, PSMD8, GSS, CCT5, CSNK1A1, CSNK1A1L, CD97, MARCKSL1, DNASE1L1,ALDH9A1, RPL34, LMAN1, FASN, CCT3, TUFM, ALDH7A1, AARS, SARS, PSMB3,PSMB2, THBS3, ACADVL, TMED10, HINT1, RGS19, GSK3B, NT5C2, GMPS, GNAQ,GNG10, MMP14, SLC26A2, SERPINB8, SERPINB9, SERPINH1, PDLIM4, VASP, DNM2,BCAM, CCT8, ANXA11, RAB5C, RAB7A, RAB13, RAB27A, PLCD1, DUSP3, BCAP31,TPMT, CAV2, PLXNA3, VAMP7, ADCY7, AKR1D1, LUM, RAP1GDS1, SLC7A2, SMS,EFNB2, STC1, THOP1, CAPZA1, BLVRA, ARFIP1, ACLY, PGGT1B, COPB1, COPA,SLC5A3, SLC16A1, IST1, SUB1, RARS, CACNA2D1, YARS, USP14, HSPA2, BCAT1,ATP12A, ATP1B3, RAD23B, EPHB4, GAS1, ALDH18A1, NAPA, MFAP2, EIF5,SLC12A2, CSE1L, VCP, ADK, LAMB2, CDH11, CDH13, SEC13, HNRNPH2, EIF3B,FCGRT, BID, ITGA1, EIF6, ANTXR2, CD81, TPI1, ACTB, EIF4A1, RPS20, PRPS1,S100A10, CDC42, DSTN, RAB8A, SPCS3, RAB2A, RAB5B, RAB10, UBE2D3, UBE2D2,UBE2M, UBE2N, RAB14, ACTR3, ACTR2, ACTR1A, COPS2, RAP1B, RAP2B, RPS3A,RPL15, RPL27, RHOA, VBP1, STXBP1, UFM1, NUTF2, HNRNPK, YWHAG, RPS7,PPP1CA, PPP1CB, NCS1, PSMC1, PSMC5, RPS8, YWHAE, RPS14, RPS18, RPS29,RPS11, SNRPE, LSM3, TMSB4XP4, TMSB4X, ARF6, PSMC6, RPL7A, ETF1, CNBP,RPS4X, RPS4Y1, RPS4Y2, PPP2CB, ACTA2, ACTG2, RHOB, RPS6, HIST1H4A,RPL23, RAP1A, RPS25, RPS26P11, RPS26, RPS28, GNB1, GNB2, RPL10A, RPL11,PPIA, FKBP1A, RPS27A, UBB, UBC, UBA52, UBBP4, GRB2, RAC1, AP2B1, GNAS,GNAS, GNAS, GNAI1, YWHAZ, PPP2R2A, PPP2R2D, DYNLL1, DYNLT1, GNG5, RPS21,GNB2L1, ACTG1, TMSB10, PPP2CA, YBX1, CSNK2B-LY6G5B-1181, CSNK2B, TPM4,ACTC1, ACTA1, ACTG2, UBE2L3, EEF1A1, EEF1A1P5, TUBA1B, KLK9, TUBB4B,PAFAH1B2, HBB, SIRPA, SIRPB1, PIP4K2B, CSNK1G2, GSTO1, ADAM17, SRPX,BASP1, DCD, SMAD3, ARF1, ARF3, ARF5, RHOG, MXRA7, TNFAIP6, DAB2, HSPG2,EFNB1, ATP8B2, HDLBP, CDK6, CDK5, CLTC, FKBP3, HNRNPU, SPTBN1, SET,FABP5, CAP1, CAP1, SLC7A5, PFKP, OCRL, PLCB3, ROR2, TAGLN, DSG1, MAP2K1,TEK, FKBP4, NUCB1, RPL6, AKAP12, GNA12, CAV1, TNFAIP2, PLAUR, GBE1,NOTCH2, GLO1, ACVR1, YWHAH, PLP2, PRKCD, PTPN11, GFPT1, FMOD, PRDX1,C1QBP, CKAP4, ENPEP, COL16A1, SPAG1, BAX, LRP1, ARHGAP1, TGM3, DHX9,CRYZ, LGALS3BP, LOXL1, MFGE8, MFGE8, DSC1, SPOCK1, VAC14, AHNAK, SMAGP,GALNT2, AP1B1, BST2, ST3GAL1, SCRN1, KIAA0196, TWF1, ASPH, EFEMP1,FSTL1, STX4, DPYD, FAP, AIMP1, ILF3, PTPRJ, DLG1, MYO1E, PTP4A2, ABR,ARHGAP5, STRN3, STRN4, FLII, COASY, SPP2, PRKAA1, PPFIA1, PPFIA3,PPFIA2, PPFIA4, PAK2, PSMD2, DNAJC3, PAPPA, PTK7, SGCG, SLC14A1, EIF3I,PLD1, DYNC112, BTN1A1, ILK, SNTB2, SLC39A6, PDAP1, ADAMS, ROCK1, TCIRG1,PICALM, TUBB3, CAMK2G, CAMK2B, NAE1, CUL1, CUL2, CUL3, CUL4A, RAB31,RAB32, TPBG, FHL1, FHL3, ALCAM, PKP1, BMPR2, TUBB2A, TUBB2B, IDI1,PRKG1, CKAP5, COTL1, SCARB2, NID2, DAG1, DSG2, SCRIB, TTLL12, DPYSL3,DYNC1H1, CTTN, FLOT2, FLNC, FZD2, GNA13, GAMT, GALE, LRRC32, FAT1, DSC3,INPP5A, TRIP12, GANAB, RFTN1, MVP, LASP1, PTGR1, RAB39A, KPNB1, NAA25,VEPH1, CHMP4A, PSMD6, Sep-02, SNX17, RAB3GAP1, SLC39A14, KARS, EIF4H,POSTN, PCOLCE, PLCB4, PLEC, PPA1, STK38, PTPRK, RASA2, RAB35, LLGL1,PCBP1, RHEB, RSU1, TGFBI, TRIP10, TRIPE, MAPRE1, MYLK, SLC1A5, SMAD1,STXBP2, ZNF14, VAMP3, VAMP2, ATP6AP1, RAB11B, RAB11A, ZYX, ADRM1, CCDC6,UAP1, IGFBP7, LAMA4, EXT1, PSMD5, PKN2, DDB1, DPYSL2, SYPL1, SGCB, ECM1,DBN1, PTGIS, FSCN1, ATP2B3, CA9, MEP1A, DDR2, UGP2, TMEM132A, INF2,KIF26B, QRICH1, LEPRE1, DAK, SERPINB12, TUBB8, PREPL, TBC1D10B, ANO6,SVEP1, TMEM119, FNDC1, RFTN2, TP5313, PLEKHO1, ALF, STON1, CYBRD1,NAALADL2, HSP90AB4P, HSP90AB2P, HERC4, TMEM67, DPCD, VPS16, COLEC12,DNMBP, LDLRAP1, OTUD7B, OTUD7A, WASF1, TPRG1L, SH3BGRL3, SLC39A1, UBR4,C9orf64, KPRP, XP32, TM9SF3, OGFRL1, SPO11, STRIP1, STRIP2, FAM171A1,BROX, FAM208B, PEAR1, ARHGEF2, RRAGB, RRAGA, RAB18, CD276, STEAP3,HGSNAT, MBLAC2, ARHGAP17, NXN, VASN, PTRHD1, LAMTOR1, TWF2, RAB12,TENM4, PTRF, CC2D1A, FAHD2A, FAHD2B, CNNM4, FAM171B, TLDC1, RHBDF2,TRAF7, CSPG4, MOXD1, LRSAM1, HHIPL2, PI16, FAT4, PACS1, CD109, C1QTNF1,FAM65A, ANKRD13D, RASSF6, UBN2, LHFPL2, VPS13C, MOB3C, UBE2R2, TUBA1A,TUBA1C, TUBA3C, TUBA3E, TUBAE, CCDC80, BTN2A1, SND1, BZW1, EIF3M,CYFIP1, TAOK1, MOB1B, CHMP1B, ZC3HAV1, NEGR1, LIMS2, GOLGA7, PODN,SRGAP1, HUWE1, TMEM179B, AMIG02, TMEM55B, TTC7B, PHLDB1, TXNDC5,SERINC5, VPS36, CAND1, TMEM200A, PPFIBP1, SBF2, SBF1, TICAM2, TMED7,TICAM2, XYLT1, STX12, AEBP1, PLD3, LIX1L, AHNAK2, CCDC50, SLITRK4,SLC44A2, SLC44A2, WDFY1, TEX2, FAM114A1, DCUN1D3, LRRC8A, SULF1, UBR1,UEVLD, CMIP, EXOC8, SLC6A16, ANKRD13A, UNC5B, SPG20, LRRC47, LYPD1, DOS,SLC35F6, C2orf18, ARHGAP18, ASCC3, EHBP1L1, PLCD3, SFRP1, MINK1, FAM26E,SLC15A4, ADAMTSL1, CCNYL1, ENAH, BMPER, DCBLD1, TDRD5, ATP11C, GOLM1,FAM63B, FAM63A, SERBP1, APOA1BP, CCNY, LRRN4CL, MAPK1IP1L, APPL2,GPRC5A, STXBP6, TSPAN14, SVIP, VANGL1, WDR48, NDNF, UBA3, TMEM167A,SPPL2B, HM13, CD99L2, PLEKHO2, NEK7, MICAL1, DTD1, IPO4, PPP4R1,UBASH3B, LRRC15, FRS2, RAB2B, PPP1R13L, CEMIP, PDCD6IP, UBTD2, FBLIM1,BRK1, SLC38A5, PHLDA1, SNX33, UBLCP1, APH1B, Clorf85, SLC44A1, PALLD,TTN, OVCA2, DDX1, PIEZO1, SLC7A6, GBF1, NCSTN, TM9SF4, NDRGL HSPH1,GCN1L1, PXDN, SGCD, PVRL2, PVRL2, RABGGTA, HTRA1, ARPC1A, STAM, HAS2,GGH, NEO1, OSTF1, GLG1, UPF1, COPS5, RAB8B, RIT1, TNPO1, NINE, DVL3,USP9X, USP9Y, CUL5, LPP, PTP4A1, SCAMP4, IGSF8, ERGIC1, SYAP1, FERMT2,LRRC59, MRGPRF, AIDA, ARL8A, ARL8B, MOB3A, DOCK10, EFHD2, EFHD1,PPP1R14B, LIMK2, RHBDF1, CTHRC1, ISOC1, KCTD12, SHISA4, CMBL, RSPRY1,L3HYPDH, CNRIP1, DYNLL2, CPNE2, S100A16, PERP, CHMP6, PGM2, SCARF2,PDLIM5, ERO1L, GMPPA, ITCH, KIRREL, LRIGL LOXL4, DCHS1, MEGF10, EXOC2,CNDP2, SNX27, IFT74, DOCK7, LRRC7, IPO9, DCBLD2, GPR124, FMNL2, TRNT1,TMEM237, GSDMA, SLC38A2, VPS35, PURB, PANX1, SNX18, NUDCD1, ERBB2IP,C16orf13, MYADM, FAM129B, PSMB7, PSMD1, PFDN5, PARK7, VAT1, S100A13,TTC1, DNAJC7, C12orf57, OSMR, CHP1, HSD17B10, NAP1L4, TM9SF2, CCT7,AGPAT1, PKP2, SH3GL1, GDF15, ARPC5L, PDCD1LG2, TMEM47, CORO1B, CPPED1,VPS25, MXRA8, SDF4, MIEN1, PELO, ERP44, LXN, ESYT1, MARVELD1, CCM2,COPS4, DCTN5, LRRC1, TUBB6, PDCD10, BDH2, NTMT1, TMED9, ACAT2, JAM3,RAB11FIP5, TBC1D10A, NAA15, SLC12A9, AP1M1, ITPA, CADM1, FRMD8, ULBP3,ULBP2, FAM129A, ZDHHC5, TTYH3, TINAGL1, C20orf27, TOLLIP, ARL6, ITFG3,WNT5B, MESDC1, EHD4, Clorf21, TRIOBP, SLK, TAOK3, SLC12A5, SLC12A7,C11orf68, TXNIP, ACBD3, UNC45A, DNAJC5, CHMP4B, RNPEP, SMOC1, EPB41L1,GLIPR2, EHD1, CDCP1, CLMP, EPS8L2, ANTXRL SH2D4A, DOCKS, ATP13A3, UBE2Z,FAM188A, MOB1A, GORASP2, C6orf211, LRRC40, SLC52A2, PPCS, UBTD1, SMURF2,GNB4, BCL2L12, CACYBP, RRAGC, RRAGD, PARVB, PROK2, GLOD4, ANKH, VAT1L,EPB41L5, EPB41L4B, CSNK1G1, CD248, S100A14, MYO10, NRXN3, VTA1,TNFRSF12A, IL1RAP, RIC8A, PLCB1, TIGAR, HINT3, NIT2, PVRL3, MYO5C,PDLIM7, SAR1A, NANS, MXRA5, SLC17A5, OLFML3, OSTC, CDC42SE1, HEBP1,VPS45, PHPT1, SERINC1, ARHGAP35, PLSCR3, TMEM256-PLSCR3, LANCL2, STARD5,ATG3, ECHDC1, LIN7C, FAM49B, NUDT15, EXOC1, TMEM30A, EVA1B, TMEM51,COMMD8, RAB20, CMTM6, ARL15, ACTR10, IGF2BP1, HSPBP1, MYOF, EHD3, EHD2,CD274, FLRT3, LMCD1, MPP6, GIMPS, VAPA, SH3BP4, VPS18, DIP2B, TENM3,PTGFRN, PCDH10, RRBP1, ANKIB1, ANKFY1, ATXN10, NCDN, MRC2, CRNN, GNG12,TNFRSF10D, SPAST, GULP1, GRHPR, CTSZ, CKLF, CTNNAL1, PEF1, DNAJB4,STK39, ADD3, ABCF2, SLC23A2, TES, SUSD2, LAMTOR3, NPC1L1, CHORDC1,TMEM2, PFDN2, SLCO3A1, LNPEP, NAGK, ANXA10, DBNL, DCTN4, VPS28, LSM7,PACSIN3, TRHDE, ANGPTL2, ITGA11, CDV3, RAB21, RAB22A, PSME2, RAB23,MCTS1, ZDHHC8, SLC39A10, ASAP1, HEG1, ANKRD50, TBC1D24, CORO1C, PYCARD,NOTCH3, ICAM5, UBQLN1, SNX12, VPS4A, PCDHGC3, STUB1, PACSIN2, STX8,PLA2G2D, PSMD13, PROCR, ABCG2, COPS3, TTF2, FZD1, SLC7A11, WASF3, SHOC2,PA2G4, CHMP2B, GNE, C14orf166, RUVBL1, NUDC, CFL2, ITM2B, SLC5A6,NCKAP1, EXOC6B, DIP2C, STK38L, EPB41L3, FAN1, LAMTOR2, TMA7, AP3M1,AP3M2, MEMO1, LSM2, SH3GLB1, CAB39, SBDS, PPIL1, UFC1, RTCB, FBXO7,RAP2C, TLN1, DAAM1, PLXND1, IRS2, LOXL2, RNF114, PPME1, PCDHGB7,PCDHGB6, PCDHGB5, PCDHA9, PHLDA3, GMPPB, TRPV2, CDC42BPB, IER3IP1, SNX9,SNX8, HEBP2, PSAT1, F11R, GPC6, NPTN, DKFZp566H1924, COPG1, LHFP, CLIC4,CFAP20, EMILIN1, DYNC1LI1, EPN1, CSNK1G3, SLC30A1, SLC4A7, ROBO1,SLC4A4, FCGBP, PCLO, CAPN7, WASF2, and combinations thereof.

In some cases, proteins that contribute to therapeutic efficacy or thatmediate paracrine signaling to effect therapeutic efficacy of exosomesisolated using CD39 binding agents comprise CD39, CD39L3, and/or CD73,or structural or functional analogues thereof differing in sequence,length or sequence and length, but retaining a collective ability toreduce ATP or ADP to the free purine adenosine. In some cases, proteinsthat contribute to therapeutic efficacy or that mediate paracrinesignaling to effect therapeutic efficacy comprise CD39. In some cases,proteins that contribute to therapeutic efficacy or that mediateparacrine signaling to effect therapeutic efficacy comprise CD39L3. Insome cases, proteins that contribute to therapeutic efficacy or thatmediate paracrine signaling to effect therapeutic efficacy compriseCD73. In some cases, exosomes disclosed herein deliver one or more ofthe above proteins to reduce inflammation. Some exosomes deliverproteins that affect local ATP, ADP and/or AMP concentrations, forexample by catalyzing the transformation of ATP into ADP, AMP or freepurine, so as to reduce at least one of ATP concentration, ADPconcentration and AMP concentration. Alternately or in combination, someexosomes deliver proteins that increase free purine concentration, forexample by converting at least one of ATP, ADP and AMP to free purine.Conversion of ATP, ADP and AMP to free purine is accomplished in somecases through a combination of proteins comprising CD39 and CD73.Alternately, in some cases a single enzymatic activity releases freepurine from at least one of ATP, ADP and AMP. Some exosomes deliver anenzyme having at least one of ATPase, ADPase, AMPase, and adenosinedepurinase activity. In various embodiments, exosomes comprise not morethan 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100or more than 100 proteins from the above list. In various embodiments,exosomes comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, 100 or more than 100 proteins from the above list.In various embodiments, exosomes comprise not more than 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, or 100% of the proteins from the above list. In variousembodiments, exosomes comprise not less than 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or100% of the proteins from the above list. In various embodiments, notmore than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the proteins from theabove list are purified, for example from exosome compositions, fortreatment of disease. In various embodiments, not less than 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 99%, or 100% of the proteins from the above list are purified,for example from exosome compositions, for treatment of disease. In somecases, purified proteins from the above list are combined with exosomecompositions, for example using the exosomes as a delivery mechanism,for treatment of disease.

Some exosome compositions isolated using CD39 binding agents disclosedherein comprise nucleic acids, such as miRNAs that contribute totherapeutic efficacy, or that mediate paracrine signaling to effecttherapeutic efficacy. In some aspects, the composition comprisesmicroRNAs (miRNAs). miRNAs include but are not limited to let7b miRNA.In some aspects, exosomes-derived miRNAs are delivered to lymphocytes toinduce a regulatory phenotype. In some aspects, the miRNAs are deliveredto lymphocytes to induce a regulatory Foxp3+ phenotype. In some aspects,the miRNAs are delivered to macrophage to induce a regulatory M2macrophage phenotype. In some aspects, the miRNA let-7b is delivered tomacrophage to induce a regulatory M2 macrophage phenotype. In someaspects, the composition comprises at least one category of moleculesuch as exogenous RNAs, proteins, antigens and microRNAs that produce animmune-stimulatory, pro-inflammatory response from a host immune system.In some aspects, the composition comprises exogenous stimulatoryantigens that produce an immune-stimulatory, pro-inflammatory responsefrom endogenous tumor infiltrating lymphocytes (TILs) in a liquid orsolid tumor. In some aspects, the composition comprises exogenousstimulatory antigens that produce an immune-stimulatory,pro-inflammatory response from endogenous tumor infiltrating lymphocytes(TILs) in a liquid or solid tumor in concert with inhibitors oflymphocyte checkpoint pathways. In some aspects, the compositioncomprises exogenous stimulatory antigens that produce an enhancedco-stimulatory, pro-inflammatory response from endogenous tumorinfiltrating lymphocytes (TILs) in a liquid or solid tumor, in concertwith antibody, bi-specific antibody or chemical inhibitors of lymphocytecheckpoint pathways such as CTLA4, PD1, PDL1, LAG3, PDL2 or CD39pathways. In some aspects, the composition comprises exogenousstimulatory antigens that produce an immune-stimulatory,pro-inflammatory response from exogenous engineered T lymphocytestargeted to a liquid or solid tumor. In some aspects, the compositioncomprises exogenous stimulatory antigens that produce animmune-stimulatory, pro-inflammatory response from exogenous dendriticcells (DCs) targeted to a specific solid tumor antigen. In some aspects,the composition comprises exogenous co-stimulatory antigens that producean immune-stimulatory, pro-inflammatory response from exogenousengineered natural killer (NK) cell targeted to a solid or liquid tumor.

A therapeutically active exosome composition such as a paracrinesignaling exosome composition disclosed herein, in some cases, comprisesan immunosuppressive drug. Immunosuppressive drugs contemplated hereininclude but are not limited to a glucocorticoid, a cytostatic, anantibody, an immunophilin inhibitor, ciclosporin, tacrolimus, sirolimusand interferon, an opioid, a TNF binding protein, a cyclooxygenaseinhibitor, an antihistamine, an antimetabolite, folic acid,methotrexate, a purine analogue, a pyrimidine analogue, a proteinsynthesis inhibitor, mycophenolate, a cytotoxic antibiotic, a steroid,an anti-TNF antibody, a TNF inhibitor, and an NSAID. Some exosomecompositions comprise anti-TNF antibodies, including but not limited toinfliximab, adalimumab, certolizumab, and golimumab. Exosomecompositions comprise TNF inhibitors including but not limited toetanercept, xanthine derivatives, and bupropion. In some cases, exosomecompositions comprise at least one NSAID. In some cases, exosomecompositions comprise at least one steroid.

Methods of Treatment

SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions isolated based on expression of CD39 disclosed hereininclude therapeutic compositions for methods and uses in treatment ofdisease, in some cases via paracrine signaling activity. Some SDC2+ andSDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions modulate an inflammation response in a mammal. Therefore,disclosed herein are methods of modulating an inflammation response in amammal by administering at least one purified SDC2+ or SDC2+CD39+stromal stem cell and SDC2+ or SDC2+CD39+ exosome compositions to thesite of the inflammation response. Also disclosed herein are purifiedSDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions for use in modulating an inflammation response in a mammalby administering at least one purified SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition to the site of theinflammation response. Also disclosed herein are purified SDC2+ andSDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions for use in preparation of a medicament for modulating aninflammation response in a mammal by administering at least one purifiedSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition to the site of the inflammation response.

Methods and uses include various routes of administration suitable forreaching sites of inflammation, which vary depending on the inflammationresponse which requires treatment. Routes of administration include butare not limited to parenteral (including subcutaneous, intravenous,intra-arterial, intraosseous, intracerebral, intra-cerebroventricular,intrathecal, intramedullary, intra-articular, intramuscular, orintraperitoneal injection), rectal, respiratory or inhalation, topical,transdermal, and oral (for example, in capsules, suspensions, ortablets). For some indications, it is desirable to administer SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosed hereinvia intravenous administration. In particular, intravenousadministration is often preferred to deliver SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions orcompositions derived from SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosomes to a mammalian lung such as a human lung.

For some indications, it is desirable to administer SDC2+ and SDC2+CD39+stromal stem cell and SDC2+ and SDC2+CD39+ exosome compositions such asparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions disclosed herein via a respiratory orinhalation route using an inhalation device. An inhalation device iscapable of administering therapeutic compositions to the respiratoryairways of a patient. In halation devices include conventionalinhalation devices such as metered dose inhalers, dry powder inhalers,jet nebulizers, ultrasonic wave nebulizers, heat vaporizers, soft mistinhalers, and high efficiency nebulizers. Nebulizers, metered doseinhalers, and soft mist inhalers deliver therapeutics by forming anaerosol, which includes droplet sizes that can easily be inhaled. Apatient within the bounds of an inhalation therapy can use the aerosol.A nebulizer is able to turn a therapeutic or medication into a fineaerosol mist that is delivered to the lungs of an individual.

Nebulizers include high efficiency nebulizers. High efficiencynebulizers are inhalation devices that comprise a micro-perforatedmembrane through which a liquid solution is converted through electricalor mechanical means into aerosol droplets suitable for inhalation. Highefficiency nebulizers can deliver a large fraction of a loaded dose to apatient. In some embodiments, the high efficiency nebulizer alsoutilizes one or more actively or passively vibrating microperforatedmembranes. In some embodiments, the high efficiency nebulizer containsone or more oscillating membranes. In some embodiments, the highefficiency nebulizer contains a vibrating mesh or plate with multipleapertures and optionally a vibration generator with an aerosol mixingchamber. In some such embodiments, the mixing chamber functions tocollect (or stage) the aerosol from the aerosol generator. In someembodiments, an inhalation valve is also used to allow an inflow ofambient air into the mixing chamber during an inhalation phase and isclosed to prevent escape of the aerosol from the mixing chamber duringan exhalation phase. In some such embodiments, the exhalation valve isarranged at a mouthpiece which is removably mounted at the mixingchamber and through which the patient inhales the aerosol from themixing chamber. Still yet, in some embodiments, the high efficiencynebulizer contains a pulsating membrane. In some embodiments, the highefficiency nebulizer is continuously operating.

For some indications, diseases or disorders, it is desirable toadminister SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ andSDC2+CD39+ exosome compositions such as paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions topically (e.g., applied directly to the skin of theindividual being treated). In some cases, topical administration is usedto treat diseases of the skin. Topical administration includesepicutaneous administration. SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosome compositions for topical administration areformulated specifically to be administered to the skin. Such topicalSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions include but are not limited to solutions, lotions, creams,ointments, gels (including hydrogels or collagen gels), foams,transdermal patches, powders, pastes, and tinctures. In some cases,SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprise a hydrogel or a collagen gel.

Certain indications, diseases, or disorders benefit from administrationof SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+exosome compositions such as paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell and SDC2+ and SDC2+CD39+ exosome compositions to theeye (e.g., intraocular or ophthalmic). SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome compositions for administration tothe eye comprise formulations (e.g., buffers or excipients) suitable foradministration to the eye.

Injection of SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ andSDC2+CD39+ exosome compositions such as paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions to a subject is effective in treating certain indications,diseases, or disorders. Delivery of an SDC2+ and SDC2+CD39+ stromal stemcell and SDC2+ and SDC2+CD39+ exosome composition via injection includesbut is not limited to injection to the lymph nodes, subcutaneousinjection, intramuscular injection, intravenous injection,intraperitoneal injection, intrathecal injection, intradermal injection,intraarticular injection, and other injection methods suitable formethods herein. SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ andSDC2+CD39+ exosome compositions for injection comprise formulations orphysiologically acceptable buffers or excipients for injection.

Certain indications, diseases, or disorders benefit from administrationof SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+exosome compositions such as paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell and SDC2+ and SDC2+CD39+ exosome compositions directlyto the heart. Direct cardiac application of SDC2+ and SDC2+CD39+ stromalstem cell and SDC2+ and SDC2+CD39+ exosome compositions such asparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions include but are not limited tointra-cardiac, intra-pericardial, or intra-coronary artery injection.

SDC2+ and SDC2+CD39+ exosome compositions such as paracrine signalingSDC2+ and SDC2+CD39+ exosome compositions used in methods of treatmentand uses in a mammal, in some cases, include one or more antigens. Inmethods of treatment and uses where the SDC2+ and SDC2+CD39+ exosomecomposition includes one or more antigens, the antigen is not exposed tothe individual's humoral immune system. In these cases, the individualdoes not develop a humoral immune response to the antigen.

SDC2+ and SDC2+CD39+ stromal stem cell and SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell and SDC2+ and SDC2+CD39+ exosome compositions used in methodsof treatment and uses in a mammal comprise SDC2 and/or CD39 or are SDC2+and SDC2+CD39+. When the stromal stem cell or exosome composition isanalyzed, at least 20% of the stromal stem cells or exosomes compriseSDC2 and at least 20% of the stromal stem cells or exosomes compriseCD39. In some cases, the SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+and SDC2+CD39+ exosome composition used in methods of treatment and usescomprise a composition where at least 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 99%, or more of the stromal stem cells or exosomes comprise SDC2and at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more of thestromal stem cells or exosomes comprise CD39. The proportion of stromalstem cells or exosomes in the composition comprising SDC2 and CD39 isdetermined by immunofluorescence, for example flow cytometry,fluorescence microscopy, electron microscopy, or other suitable method.

Stromal cell compositions used in methods of treatment herein, in somecases, are modified to increase the therapeutic efficacy of the stromalstem cell composition. In some cases, cells are genetically modified tooverexpress an apyrase as disclosed herein, such as CD39, a structuralor functionally active variant of CD39 or other protein having apyraseactivity. In some cases, cells are genetically modified to overexpressCD39L3. Genetic modification of stromal cells is accomplished by methodsincluding but not limited to transfection of the stromal cells with oneor more plasmids that comprise the CD39 or CD39L3 coding sequence and apromoter, such as a CMV, SV40, EF1a, or CAG promoter. Geneticmodification of stromal cells, in some cases, is accomplished byinfection of the stromal cells with a virus that comprises the CD39 orCD39L3 coding sequence and a promoter.

While SDC2+ and SDC2+CD39+ exosome compositions such as paracrinesignaling SDC2+ and SDC2+CD39+ exosome compositions described herein, insome cases, are derived from cells isolated based on expression of CD39,the exosome compositions do not comprise living cells.

The exosome compositions, therefore, are non-tumorigenic, that is, theydo not increase the susceptibility of a subject to developing a tumor orcancer.

Methods of treatment and uses disclosed herein comprise administering toa mammal an SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition comprising SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosomes, for example paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosomes or in vitro SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosomes, and mixtures of SDC2+ and SDC2+CD39+ exosomes andSDC2+ and SDC2+CD39+ mesenchymal stem cells (e.g., SDC2+ and SDC2+CD39+mesenchymal stromal stem cells). In some instances, methods of treatmentand uses use exosome compositions that include compositions comprisingSDC2+ and SDC2+CD39+ exosomes or in vitro exosomes and regulatory Tcells. Regulatory T cells include CD25+ regulatory T cells, CD4+regulatory T cells, FoxP3+ regulatory T cells, CD25+CD4+ FoxP3+regulatory T cells, and combinations thereof. In some instances, methodsof treatment and uses that use exosome compositions include compositionscomprising in vitro SDC2+ and SDC2+CD39+ exosomes, SDC2+ and SDC2+CD39+mesenchymal stem cells, and regulatory T cells.

Methods of treatment and uses herein use SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ exosomecompositions that retain potency or activity after being frozen orcryopreserved without the use of a cryoprotectant. Cryoprotectantsinclude DMSO, glycerol, polyethylene glycol, propylene glycol,glycerine, polyvinylpyrolidone, sorbitol, dextran, and trehalose. Theexosome compositions used in methods of treatment and uses also retainpotency after being frozen without using special freezing protocols.Special freezing protocols include flash freezing, programmable ratefreezer, and freezing in an insulated container. The exosomecompositions used for methods of treatment and uses are frozen in bufferor culture media. Buffers include physiologically acceptable bufferssuch as phosphate buffer, histidine buffer, citrate buffer, acetatebuffer, and other suitable buffers. In some cases, methods of treatmentand uses herein use exosome compositions are lyophilized or have beenlyophilized.

Methods of treatment and uses disclosed herein use SDC2+ and SDC2+CD39+stromal stem cell compositions that retain potency or activity afterbeing frozen or cryopreserved. Cryoprotectants include DMSO, glycerol,polyethylene glycol, propylene glycol, glycerine, polyvinylpyrolidone,sorbitol, dextran, and trehalose. The SDC2+ and SDC2+CD39+ stromal stemcell compositions used in methods of treatment and uses also retainpotency after being frozen using special freezing protocols. Specialfreezing protocols include flash freezing, programmable rate freezer,and freezing in an insulated container. The SDC2+ and SDC2+CD39+ stromalstem cell compositions used for methods of treatment and uses are frozenin buffer or culture media mixed with a cryoprotectant. Buffers includephysiologically acceptable buffers such as phosphate buffer, histidinebuffer, citrate buffer, acetate buffer, and other suitable buffers.

Methods of treatment and uses disclosed herein comprise administrationof compositions comprising SDC2+ stromal stem cells isolated based onexpression of CD39 in a therapeutically effective amount. Administrationof a therapeutically effective amount of SDC2+ and SDC2+CD39+ stromalstem cells, in some cases, comprises administration of 10{circumflexover ( )}3 to 10{circumflex over ( )}8 SDC2+ and SDC2+CD39+ stromal stemcells, for example 10{circumflex over ( )}3, 10{circumflex over ( )}4,10{circumflex over ( )}5, 10{circumflex over ( )}6, 10{circumflex over( )}7, 10{circumflex over ( )}8, or more SDC2+ and SDC2+CD39+ stromalstem cells of the composition. Methods of treatment and uses includeadministration of SDC2+ and SDC2+CD39+ stromal stem cells compositions,which in some cases, are concentrated to be diluted by the individual orhealth care provider prior to administration. In some cases, methods oftreatment and uses comprise administration of SDC2+ and SDC2+CD39+stromal stem cells compositions that are diluted and ready to beadministered by the individual or health care provider. In some cases,methods of treatment and uses comprise administration of SDC2+ andSDC2+CD39+ stromal stem cells compositions that are contained in singleuse vials, syringes, or IV bags. In some cases, methods of treatment anduses comprise administration of a dose from a container comprisingmultiple doses.

Methods of treatment and uses disclosed herein comprise administrationof compositions comprising exosomes such as paracrine signaling exosomesin a therapeutically effective amount. Administration of atherapeutically effective amount of exosomes, in some cases, comprisesadministration of 10⁶-10⁸ exosomes, for example 10⁶, 10⁷, 10⁸, or moreexosomes of the composition. In some cases, administration of atherapeutically effective amount of exosomes comprises administration of1 mg to 700 mg of exosomes, for example 1 mg, 10 mg, 20 mg, 50 mg, 100mg, 150 mg, 200 mg, 250 mg, 500 mg, 750 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 10 mg, 20 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500mg, 600 mg, 700 mg, or more exosomes in the composition. Methods oftreatment and uses include administration of exosome compositions, whichin some cases, are concentrated to be diluted by the individual prior toadministration. In some cases, methods of treatment and uses compriseadministration of exosome compositions that are diluted and ready to beadministered by the individual. In some cases, methods of treatment anduses comprise administration of exosome compositions that are containedin single use vials or syringes. In some cases, methods of treatment anduses comprise administration of a dose from a container comprisingmultiple doses.

Methods of treatment and uses disclosed herein comprise administrationof SDC2+ and SDC2+CD39+ exosome compositions such as paracrine signalingSDC2+ and SDC2+CD39+ exosome compositions comprising proteins with orwithout therapeutic efficacy. Proteins include but are not limited toIL-12, suppressor of cytokine signaling (SOCS), p53, PTEN, CD52, TSC1,FOXP3, Soluble Immune Response Suppressor (SIRS), TGFB, CD39, CD39L3,CD73, and Maspin. Some methods and uses comprise administration of anenzyme having ATPase, ADPase, AMPase, or adenosine depurinase activity.

Methods of treatment and uses herein, in some cases, compriseadministration of a therapeutically active SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome composition such as paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition that comprises an immunosuppressive drug.Non-limiting examples of immunosuppressive drugs used in SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for treatment include but are not limited to aglucocorticoid, a cytostatic, an antibody, an immunophilin inhibitor,ciclosporin, tacrolimus, sirolimus and interferon, an opioid, a TNFbinding protein, a cyclooxygenase inhibitor, an antihistamine, anantimetabolite, folic acid, methotrexate, a purine analogue, apyrimidine analogue, a protein synthesis inhibitor, mycophenolate, acytotoxic antibiotic, a steroid, an anti-TNF antibody, a TNF inhibitor,and an NSAID. SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions for methods of treatment and usescomprise anti-TNF antibodies, including but not limited to infliximab,adalimumab, certolizumab, and golimumab. Methods of treatment and usescomprise administration of SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosome compositions that comprise TNF inhibitorsincluding but not limited to etanercept, xanthine derivatives, andbupropion. SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions for methods of treatment and usesherein, in some cases, comprise NSAIDs. In some cases SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for methods of treatment and uses comprise one or moresteroids.

Inflammatory diseases and disorders of the immune system result becauseof an over activation or inappropriate activation of the immune system.One or more cell types in the immune system may contribute toinflammatory and immune disease, for example CD4+ helper T cells, CD8+cytotoxic T cells, Th17 cells, dendritic cells, macrophages, mast cells,leukocytes, neutrophils, eosinophils, basophils, monocytes, andcombinations thereof. Often these cells synergize and amplify theinflammatory response through cell derived mediators such as enzymes,cytokines, chemokines, and other immune mediators. Immune mediatorsinclude but are not limited to lysosome granules, histamine, IFNγ, IL-8,leukotriene B4, nitric oxide, prostaglandins, TNFα, IL-1, IL-10, IL-17,IL-2, and combinations thereof. Acute inflammation is a necessarycomponent of the immune response to disease; however, chronicinflammation generally leads to inflammatory disease resulting in tissuedestruction by inflammatory cells. In some cases, inflammation iscaused, at least in part, by increased extracellular ATP at the site ofinflammation. In some cases, SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosome compositions such as paracrine signalingSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, reduce extracellular ATP thereby reducinginflammation. In some cases, inflammation is caused, at least in part,by increased extracellular ADP at the site of inflammation. In somecases, SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome compositions such as paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosedherein, reduce extracellular ADP thereby reducing inflammation. In somecases, inflammation is caused, at least in part, by increasedextracellular AMP at the site of inflammation. In some cases, SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosed herein,reduce extracellular AMP thereby reducing inflammation. In some cases,inflammation is caused, at least in part, by increased extracellularadenosine at the site of inflammation. In some cases, SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosed herein,reduce extracellular adenosine thereby reducing inflammation. In somecases, SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome compositions, such as paracrine signaling compositions reduceinflammation by delivery of at least one enzyme having ATPase, ADPase,AMPase, or adenosine depurinase activity. In some cases, SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions, such as paracrine signaling compositions reduceinflammation by delivery of at least one of or both of the CD39 and CD73enzymes, or structural or functional analogues thereof differing insequence, length or sequence and length, but retaining a collectiveability to reduce ATP or ADP to the free purine adenosine in theextracellular space. While inflammatory diseases present with a varietyof symptoms that depend on the tissue or organ affected by inflammation,some commonalities include pain, heat, redness, swelling, and loss oftissue or organ function. In some cases, SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions prevent orreverse all or some of the above inflammatory responses.

Disclosed herein are methods of modulation of an inflammatory responsein a mammal using SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions. Also disclosed are SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions for usein modulation of an inflammatory response in a mammal. Also disclosedare SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome compositions for use in preparation of a medicament formodulation of an inflammatory response in a mammal. An inflammatoryresponse, in some cases, is an immune response. In some cases, aninflammatory response is an autoimmune response. Immune responses,inflammatory responses, and autoimmune responses, often lead to thedevelopment of diseases or disorders in need of treatment in a mammal.Diseases and disorders caused by an immune response, inflammatoryresponse, or autoimmune response affect nearly every tissue of the bodyand are generally characterized by an overactive or otherwiseinappropriate response by the immune system of a mammal. In some cases,the immune response, inflammatory response, or autoimmune response isfrom the adaptive immune system. In some cases, the immune response,inflammatory response, or autoimmune response is from the innate immuneresponse. In some cases, the immune response, inflammatory response, orautoimmune response results in excess secretion of cytokines, cytotoxicT cell activity, antibody production, T cell proliferation, swelling,redness, fever, edema, or other response by a cell or tissue of theimmune system. In some cases the immune response is mediated oralleviated by administration of an SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as a paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition.

Disclosed herein are methods of treatment of an inflammation response orimmune response comprising administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition. Alsodisclosed herein are SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions for use in treatment of an inflammationresponse or immune response. Also disclosed herein are SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for use in preparation of a medicament for treatment of aninflammation response or immune response. The response includes but isnot limited to sepsis, acute respiratory distress syndrome (ARDS),Crohn's disease, inflammatory bowel syndrome, rheumatoid arthritis,osteoarthritis, graft versus host disease, multiple sclerosis,amyotrophic lateral sclerosis, motor neuron disorders, Sjogren'ssyndrome, non-healing dermal wounds, bone fractures, concussion wounds,burns, cachexia, sarcophenia, Dermatomyositis, Fibromyalgia, Inclusionbody myositis, Myositis, Myasthenia gravis, Neuromyotonia,Paraneoplastic cerebellar degeneration, Polymyositis, and combinationsthereof. In some cases, the inflammatory response comprises aninflammatory liver disease. In some cases the inflammation response ismediated or alleviated by administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition such as aparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition.

An inflammatory response in need of treatment using SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosedherein, in some cases, comprises a diabetic complication. Diabeticcomplications are often seen in individuals with diabetes. In somecases, the diabetic complications occur in individuals with type 1diabetes. In some cases, the diabetic complications occur in individualswith type 2 diabetes. Diabetic complications include, but are notlimited to atherosclerosis, nephropathy, cardiomyopathy, neuropathy, akidney disorder, kidney failure, diabetic ulcers, leg ulcers, and otherconditions occurring in patients with diabetes. In some cases thediabetic complication is mediated or alleviated by administration of aSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition such as a paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or and SDC2+CD39+ SDC2+ and SDC2+CD39+ exosome composition.Upon SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome administration, symptoms of the diabetic condition areameliorated, such that some function is regained in some cases. Often,these benefits are observed in diabetic individuals without impactingblood glucose levels.

SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, in some cases, are useful for treating oralleviating type 2 diabetes, also known as noninsulin-dependent diabetesmellitus or adult onset diabetes. Type 2 diabetes is associated withincreased systemic inflammation which reduces sensitivity to insulin inpatients with type 2 diabetes. In some cases, systemic inflammation isassociated with obesity, heart disease, atherosclerosis and metabolicsyndrome. Symptoms of type 2 diabetes include but are not limited tofatigue, hunger, non-healing sores, heart disease, stroke, diabeticretinopathy, blindness, kidney failure, reduced blood flow, hyperosmolarhyperglycemia, and combinations thereof. Reduction of inflammation, byanti-inflammatory drugs or compositions, such as SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosedherein, reduce one or more symptoms and in some cases cure type 2diabetes. In particular, SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+and SDC2+CD39+ exosome administration in some cases amelioratesdiabetes-associated kidney damage, in some cases without impactingglucose levels in the exosome recipient.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein comprises a heart disorder. Heartdisorders include but are not limited to myocarditis, postmyocardialinfarction syndrome, postperiocardiotomy syndrome, and subacutebacterial endocarditis. In some cases the heart disorder is mediated oralleviated by administration of an SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as a paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein comprises a kidney disorder. Kidneydisorders include but are not limited to anti-glomerular basementmembrane nephritis, interstitial cystitis, and lupus nephritis. Somekidney disorders result from or are associated with diabetes such astype 1 diabetes or type 2 diabetes. In some cases the kidney disorder istreated, mediated or alleviated by administration of an SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition. An example of an SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosome composition mediating a kidney disorder isa paracrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+and SDC2+CD39+ exosome composition. Upon SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome administration, symptoms ofthe kidney condition are ameliorated, such that some kidney function isregained in some cases. Often, these benefits are observed in diabeticindividuals without impacting blood glucose levels.

Some inflammatory responses in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions herein comprise a liver disorder. Liver disorders includebut are not limited to autoimmune hepatitis, Primary biliary cirrhosis,and Primary sclerosing cholangitis. In some cases the liver disorder ismediated or alleviated by administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition such as aparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein comprises a lung disorder. Lung disordersinclude but are not limited to acute respiratory distress syndrome(ARDS), Antisynthetase syndrome, asthma, chronic obstructive pulmonarydisease, cystic fibrosis, atelectasis, bronchitis, emphysema, pneumonia,and pulmonary edema. In some cases the lung disorder is mediated oralleviated by administration of an SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as a paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein comprises a skin disorder. Skin disordersinclude but are not limited to Alopecia Areata, Autoimmune Angioedema,autoimmune progesterone dermatitis, autoimmune urticaria, Bullouspemphigoid, Cicatricial pemphigoid, Dermatitis herpetiformis, Discoidlupus erythematosus, Epidermolysis bullosa acquisita, Erythema nodosum,Gestational pemphigoid, Hidradenitis suppurativa, Lichen planus, Lichensclerosus, Linear IgA disease, Morphea, Pemphigus vulgaris, Pityriasislichenoides et varioliformis acuta, Mucha-Habermann disease, Psoriasis,Systemic scleroderma, Vitiligo, Addison's disease, autoimmunepolyendocrine syndrome, autoimmune polyendocrine syndrome type 2, andautoimmune polyendocrine syndrome type 3. In some cases the skindisorder is mediated or alleviated by administration of an SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositionsuch as a paracrine signaling SDC2+ and SDC2+CD39+ stromal stem cell orSDC2+ and SDC2+CD39+ exosome composition.

Some inflammatory responses in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions herein, comprise a pancreas disorder. Pancreas disordersinclude but are not limited to autoimmune pancreatitis and diabetesmellitus type 1. In some cases the pancreatic disorder is mediated oralleviated by administration of an SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as a paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition.

SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, in some cases, are useful in treating oralleviating Diabetes mellitus type 1, also known as type 1 diabetes.Diabetes mellitus type 1 is caused by destruction of insulin secretingbeta cells in the pancreas, at least in part, by autoreactive T cells.Symptoms of Diabetes mellitus type 1 include but are not limited toincreased blood and urine glucose levels, polyuria (frequent urination),polydipsia (increased thirst), polyphagia (increased hunger), weightloss, diabetic ketoacidosis, nonketoic hyperosmolar coma, heart disease,stroke, kidney failure, foot ulcers, eye damage, and combinationsthereof. In the latent autoimmune or early stage of diabetes,immunosuppression or reduction of the immune response, byanti-inflammatory drugs or compositions, such as SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions disclosedherein, reverse, slow, and prevent increased destruction of the betacells, leading to a reduction in one or more symptoms, and in somecases, a cure of the diabetes mellitus type 1. In particular,administration of an SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition in some cases treats, mediates, orameliorates the symptoms of type 1 diabetes-related kidney disorders. Insome cases this beneficial effect of SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome administration is independent ofany impact on glucose levels of SDC2+ and SDC2+CD39+ stromal stem cellor SDC2+ and SDC2+CD39+ exosome administration.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, comprises a thyroid disorder. Thyroiddisorders include but are not limited to autoimmune thyroiditis, Ord'sthyroiditis and Graves' disease. In some cases the thyroid disorder ismediated or alleviated by administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition such as aparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, comprises an exocrine disorder. Exocrinedisorders include but are not limited to a reproductive organ disorder,autoimmune oophoritis, endometriosis, and autoimmune orchitis. In somecases the exocrine disorder is mediated or alleviated by administrationof an SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition such as a paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, comprises a digestive system disorder.Digestive disorders include but are not limited to autoimmuneenteropathy, Celiac disease, Crohn's disease, microscopic colitis,inflammatory bowel disease, and ulcerative colitis. In some cases thedigestive system disorder is mediated or alleviated by administration ofan SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition such as a paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition.

Some inflammatory responses in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions herein, comprise a blood disorder. Blood disorders includebut are not limited to antiphospholipid syndrome, aplastic anemia,autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome,autoimmune neutropenia, autoimmune thrombocytopenic purpura, Coldagglutinin disease, Essential mixed cryoglobulinemia, Evans syndrome,IgG4-related systemic disease, Paroxysmal nocturnal hemoglobinuria,Pernicious anemia, Pure red cell aplasia, and Thrombocytopenia. Often,the blood disorder is mediated or alleviated by administration of anSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition, such as a paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome composition.

Often, an inflammatory response in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein comprises a connective tissue, multi-organor systemic disorder. Connective tissue, multi-organ, or systemicdisorders include but are not limited to adiposis dolorosa, Adult-onsetStill's disease, Ankylosing Spondylitis, CREST syndrome, Drug-inducedlupus, enthesitis-related arthritis, Eosinophilic fasciitis, Feltysyndrome, juvenile Arthritis, Lyme disease (Chronic), mixed connectivetissue disease, palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, psoriatic arthritis, reactive arthritis,relapsing polychondritis, retroperitoneal fibrosis, rheumatic fever,rheumatoid arthritis, sarcoidosis, schnitzler syndrome, Systemic LupusErythematosus, and undifferentiated connective tissue disease. In somecases the connective tissue, multi-organ or systemic disorder ismediated or alleviated by administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition such as aparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, comprises a nervous system disorder.Nervous system disorders include but are not limited to acutedisseminated encephalomyelitis, acute motor axonal neuropathy,anti-N-methyl-D-aspartate receptor encephalitis, Balo concentricsclerosis, Bickerstaffs encephalitis, chronic inflammatory demyelinatingpolyneuropathy, Guillain-Barré syndrome, Hashimoto's encephalopathy,idiopathic inflammatory demyelinating diseases, Lambert-Eaton myasthenicsyndrome, multiple sclerosis, pediatric autoimmune neuropsychiatricdisorder associated with Streptococcus, progressive inflammatoryneuropathy, restless leg syndrome, Stiff person syndrome, Sydenhamchorea, Alzheimer's disease, Parkinson's disease, ALS, and transversemyelitis. In some cases, the nervous system disorder is mediated oralleviated by administration of an SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as a paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition. In some cases, the nervous system disorder is acentral nervous system disorder. In some cases, administration of anSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition such as a paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome composition disclosed hereinresults in exosomes or other factors crossing the blood brain barrierthereby reducing inflammation in the brain.

Some inflammatory responses in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions herein, comprise an eye disorder. Eye disorders include butare not limited to autoimmune retinopathy, autoimmune uveitis, Cogansyndrome, Graves's ophthalmopathy, intermediate uveitis, Ligneousconjunctivitis, Mooren's ulcer, Neuromyelitis optica, Opsoclonusmyoclonus syndrome, Optic neuritis, Scleritis, Susac's syndrome,Sympathetic ophthalmia, and Tolosa-Hunt syndrome. Often the eye disorderis mediated or alleviated by administration of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition such as aparacrine signaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition.

Additional inflammatory responses in need of treatment using SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions herein, comprises an ear disorder. Ear disorders includebut are not limited to autoimmune inner ear disease and Meniere'sdisease. In some cases the ear disorder is mediated or alleviated byadministration of an SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition such as a paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition.

In some cases, an inflammatory response in need of treatment using SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein, comprises a vascular system disorder.Vascular system disorders include but are not limited to Anti-neutrophilcytoplasmic antibody-associated vasculitis, Behçet's disease,Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonlein purpura,Kawasaki's disease, Leukocytoclastic vasculitis, Lupus vasculitis,Rheumatoid vasculitis, Microscopic polyangiitis, Polyarteritis nodosa,Polymyalgia rheumatica, Urticarial vasculitis, and Vasculitis. In somecases the vascular system disorder is mediated or alleviated byadministration of an SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition such as a paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition.

Methods of treatment and uses disclosed herein comprise identificationof a patient or a group of patients for treatment by administration ofSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions. Identificationof a patient group, in some cases, is done by a physician or otherhealthcare professional. The physician or other healthcare professionaluses criteria known by those in the medical field to determine whetheran individual, patient, or group of patients is a candidate fortreatment of an inflammation, immune or autoimmune response.

Methods of Delivery

SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions, purified basedon expression of CD39, as described herein, are used in methods ofdelivery of immuno-modulatory signals to the intracellular space of amammal. SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome compositions such as paracrine signaling SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions, asdescribed herein, are also for use in delivery of immuno-modulatorysignals to the intracellular space of a mammal. Immuno-modulatorysignals comprise compositions that modulate the inflammatory or immuneresponse in a mammal. In some cases, the exosome composition is isolatedor purified from a stromal cell, a SDC2+ cell, mesenchymal stem cell, ora SDC2+ mesenchymal stem cell, wherein cells are purified based onexpression of CD39. The exosome composition contains theimmuno-modulatory signal thereby delivering the immuno-modulatory signalto the intracellular space of the mammal when administered to the mammalwithout exposing the contents of the exosome composition to the humoralimmune system of the mammal.

Once administered, delivery of the SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome composition such as paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome composition to the intracellular space of the mammal occursthough processes including but not limited to phagocytosis, endocytosis,or fusion. The SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome composition is administered to the mammal in need ofimmuno-modulatory signaling by suitable routes of administration,appropriate to the need of the mammal. Routes of administration includebut are not limited to parenteral (including subcutaneous, intravenous,intra-arterial, intraosseous, intracerebral, intracerebroventricular,intrathecal, intramedullary, intraarticular, intramuscular, orintraperitoneal injection), rectal, respiratory or inhalation, topical,transdermal, or oral (for example, in capsules, suspensions, ortablets).

Methods and uses of delivery use SDC2+ and SDC2+CD39+ stromal stem cellor SDC2+ and SDC2+CD39+ exosome compositions such as paracrine signalingSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein that are formulated in a physiologicallyacceptable buffer and at least one excipient. Non-limiting examples ofexcipients include sucrose, trehalose, polyethylene glycol, apolysaccharide, a carrier protein, an inert protein, dextran, hydroxylethyl starch (BETA), PEG-4000, gelatin, PLGA, Eudragit RS 100Nanoparticles, and combinations thereof. Such SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome compositions arestored at a temperature determined to be most stable (i.e., wherein theSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition retains highest potency). In some cases, addition of one ormore excipients allows the composition to retain potency when stored ata higher temperature than otherwise would be possible.

Methods and uses of delivery of SDC2+ and SDC2+CD39+ stromal stem cellor SDC2+ and SDC2+CD39+ exosome compositions such as paracrine signalingSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein include compositions that comprisemixtures of SDC2+ and SDC2+CD39+ exosomes and SDC2+ mesenchymal stemcells (e.g., SDC2+ mesenchymal stromal stem cells). In some instances,SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions include compositions comprising in vitro SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomes andregulatory T cells. Regulatory T cells include CD25+ regulatory T cells,CD4+ regulatory T cells, FoxP3+ regulatory T cells, CD25+CD4+ FoxP3+regulatory T cells, and combinations thereof. In some instances, SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions include compositions comprising in vitro exosomes, SDC2+mesenchymal stem cells (e.g., SDC2+ mesenchymal stromal stem cells), andregulatory T cells.

Stromal cell compositions for methods of delivery herein, in some cases,are modified to increase the therapeutic efficacy of the stromal stemcell composition. In some cases, cells are genetically modified tooverexpress CD39. In some cases, cells are genetically modified tooverexpress CD39L3. In some cases, cells are genetically modified tooverexpress an apyrase as disclosed herein, such as CD39, a structuralor functionally active variant of CD39 or other protein having apyraseactivity. Genetic modification of stromal cells is accomplished bymethods including but is not limited to transfection of the stromalcells with one or more plasmids that comprise the CD39 or CD39L3 codingsequence and a promoter, such as a CMV, SV40, EF1a, or CAG promoter.Genetic modification of stromal cells, in some cases, is accomplished byinfection of the stromal cells with a virus that comprises the CD39 orCD39L3 coding sequence and a promoter.

Methods and uses of delivery comprise administration of compositionscomprising SDC2+ and SDC2+CD39+ stromal stem cells isolated based onexpression of CD39 in a therapeutically effective amount. Administrationof a therapeutically effective amount of SDC2+ and SDC2+CD39+ stromalstem cells, in some cases, comprises administration of 10{circumflexover ( )}3 to 10{circumflex over ( )}8 SDC2+ and SDC2+CD39+ stromal stemcells, for example 10{circumflex over ( )}3, 10{circumflex over ( )}4,10{circumflex over ( )}5, 10{circumflex over ( )}6, 10{circumflex over( )}7, 10{circumflex over ( )}8, or more SDC2+ and SDC2+CD39+ stromalstem cells of the composition. Methods and uses of delivery includeadministration of SDC2+ and SDC2+CD39+ stromal stem cells compositions,which in some cases, are concentrated to be diluted by the individual orhealth care provider prior to administration. In some cases, methods anduses of delivery comprise administration of SDC2+ and SDC2+CD39+ stromalstem cells compositions that are diluted and ready to be administered bythe individual or health care provider. In some cases, methods and usesof delivery comprise administration of SDC2+ and SDC2+CD39+ stromal stemcells compositions that are contained in single use vials, syringes, orIV bags. In some cases, methods and uses of delivery compriseadministration of a dose from a container comprising multiple doses.

Methods and uses of delivery comprise administration of exosomecompositions such as paracrine signaling exosome compositions in atherapeutically effective amount. A therapeutically effective amount ofexosomes, in some cases, ranges from 10⁶-10⁸ exosomes, for example 10⁶,10⁷, 10⁸, or more exosomes in the composition. In some cases, atherapeutically effective amount of exosomes ranges from 1 mg to 700 mgof exosomes, for example 1 mg, 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 500 mg, 750 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 10mg, 20 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700mg, or more exosomes in the composition. Exosome compositions, in somecases, are concentrated to be diluted by the individual prior toadministration. In some cases, exosome compositions are diluted andready to be administered by the individual. In some cases, exosomecompositions are contained in single use vials or syringes. In somecases, multiple doses are present in a single container.

Methods and uses of delivery use SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ exosomecompositions that comprise proteins with or without therapeuticefficacy. Proteins include but are not limited to IL-12, suppressor ofcytokine signaling (SOCS), p53, PTEN, CD52, TSC1, FOXP3, Soluble ImmuneResponse Suppressor (SIRS), TGFB, CD39, CD39L3, CD73, and Maspin.

Methods and uses of delivery to an intracellular space include SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions topically (e.g.,applied directly to the skin of the individual being treated). In somecases, topical administration is used to treat diseases of the skin.Topical administration includes epicutaneous administration. SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for topical administration are formulated specifically tobe administered to the skin. Such topical SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions include but arenot limited to solutions, lotions, creams, ointments, gels (includinghydrogels or collagen gels), foams, transdermal patches, powders,pastes, and tinctures. In some cases, SDC2+ and SDC2+CD39+ stromal stemcell or SDC2+ and SDC2+CD39+ exosome compositions comprise a hydrogel ora collagen gel.

Methods and uses of delivery to an intracellular space includeadministration of SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions such as paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions to the eye (e.g., intraocular or ophthalmic). SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for administration to the eye comprise formulations (e.g.,buffers or excipients) suitable for administration to the eye.

Methods and uses of delivery to an intracellular space includeadministration by injection. Injection of SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions such as paracrinesignaling SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+exosome compositions to a subject is effective in treating certainindications, diseases, or disorders. Delivery of an SDC2+ and SDC2+CD39+stromal stem cell or SDC2+ and SDC2+CD39+ exosome composition viainjection includes but is not limited to injection to the lymph nodes,subcutaneous injection, intramuscular injection, intravenous injection,intraperitoneal injection, intrathecal injection, intradermal injection,intraarticular injection, and other suitable injection methods. SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions for injection comprise formulations or physiologicallyacceptable buffers or excipients for injection.

Methods and uses of delivery to an intracellular space includeadministration of SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions such as paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions disclosed herein via a respiratory or inhalation routeusing an inhalation device. An inhalation device is capable ofadministering therapeutic compositions to the respiratory airways of apatient. In halation devices include conventional inhalation devicessuch as metered dose inhalers, dry powder inhalers, jet nebulizers,ultrasonic wave nebulizers, heat vaporizers, soft mist inhalers, andhigh efficiency nebulizers. Nebulizers, metered dose inhalers, and softmist inhalers deliver therapeutics by forming an aerosol which includesdroplet sizes that can easily be inhaled. The aerosol can be used by apatient within the bounds of an inhalation therapy. A nebulizer is ableto turn a therapeutic or medication into a fine aerosol mist that isdelivered to the lungs of an individual.

Methods and uses of delivery to an intracellular space include use ofSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions comprising animmunosuppressive drug. Non-limiting examples of immunosuppressive drugsinclude but are not limited to a glucocorticoid, a cytostatic, anantibody, an immunophilin inhibitor, ciclosporin, tacrolimus, sirolimusand interferon, an opioid, a TNF binding protein, a cyclooxygenaseinhibitor, an antihistamine, an antimetabolite, folic acid,methotrexate, a purine analogue, a pyrimidine analogue, a proteinsynthesis inhibitor, mycophenolate, a cytotoxic antibiotic, a steroid,an anti-TNF antibody, a TNF inhibitor, and an NSAID. SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprise anti-TNF antibodies, including but not limited toinfliximab, adalimumab, certolizumab, and golimumab. SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprise TNF inhibitors including but not limited toetanercept, xanthine derivatives, and bupropion. In some cases, SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprise one or more NSAIDs. In some cases, SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprise one or more steroids.

Supplemented Stem Cell and Stromal Stem Cell Compositions

Disclosed herein are SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ andSDC2+CD39+ exosome compositions such as paracrine signaling SDC2+ andSDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions comprising supplemented stem cell and stromal stem cellcompositions, wherein SDC2+ and SDC2+CD39+ stromal stem cells areisolated based on expression of CD39. Supplemented stem cell and stromalstem cell compositions comprise cultured stem cells or stromal stemcells and at least one SDC2+ and SDC2+CD39+ stromal stem cell or SDC2+and SDC2+CD39+ exosome composition disclosed herein. In some cases, theSDC2+ and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecomposition is isolated from the cultured stem cells or stromal stemcells based on expression of CD39 and then combined with the culturedstem cells to enhance the therapeutic efficacy of the cultured stemcells.

Supplemented stem cell and stromal stem cell compositions comprise SDC2+and SDC2+CD39+ stromal stem cell or SDC2+ and SDC2+CD39+ exosomecompositions such as paracrine signaling SDC2+ and SDC2+CD39+ stromalstem cell or SDC2+ and SDC2+CD39+ exosome compositions, wherein theSDC2+ and SDC2+CD39+ stromal stem cells or SDC2+ and SDC2+CD39+ exosomesare isolated based on expression of CD39. In some compositions, at least20% of the stromal stem cells or exosomes in the composition are SDC2+or comprise SDC2. In some cases at least 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 99%, or more of the stromal stem cells or exosomescomprise SDC2. In some instances, SDC2 is found on the surface of thestromal stem cell or exosome or at least comprising a polypeptideportion that is found on the surface of the stromal stem cell orexosome. In some instances, SDC2 is found at the interior of the stromalstem cell or exosome. The proportion of SDC2+ stromal stem cell or SDC2+and SDC2+CD39+ exosomes in the composition comprising SDC2 is determinedby immunofluorescence, for example FACS, microscopy, or other suitablemethod.

Supplemented stem cell compositions herein, in some cases, comprisestromal stem cells modified to increase the therapeutic efficacy of thestromal stem cell composition. In some cases, cells are geneticallymodified to overexpress CD39. In some cases, cells are geneticallymodified to overexpress CD39L3. In some cases, cells are geneticallymodified to overexpress an apyrase as disclosed herein, such as CD39, astructural or functionally active variant of CD39 or other proteinhaving apyrase activity. Genetic modification of stromal cells isaccomplished by methods including but not limited to transfection of thestromal cells with one or more plasmids that comprise the CD39 or CD39L3coding sequence and a promoter, such as a CMV, SV40, EF1a, or CAGpromoter. Genetic modification of stromal cells, in some cases, isaccomplished by infection of the stromal cells with a virus thatcomprises the CD39 or CD39L3 coding sequence and a promoter.

Supplemented stem cell compositions comprise cultured stem cells orstromal stem cells that in some cases are SDC2+. At least 20% of thecultured stem cells or stromal stem cells are SDC2+ in some cases. Thecultured stem cell or stromal stem cell composition comprises, in somecases, 20% to 90% SDC2+ cells, for example 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% SDC+ cells or more than 90% SDC2 cells, up to and including auniform population of 100% SDC2 positive cells.

Supplemented stem cell and stromal stem cell compositions in some cases,comprise SDC2+ and SDC2+CD39+ exosome compositions such as paracrinesignaling SDC2+ and SDC2+CD39+ exosome compositions comprising proteinswith or without therapeutic efficacy. Proteins include but are notlimited to IL-12, suppressor of cytokine signaling (SOCS), p53, PTEN,CD52, TSC1, FOXP3, Soluble Immune Response Suppressor (SIRS), TGFB,CD39, CD39L3, CD73, and Maspin.

Supplemented stem cell and stromal stem cell compositions comprise, insome cases, an SDC2+ and SDC2+CD39+ exosome composition such asparacrine signaling SDC2+ and SDC2+CD39+ exosome composition comprisingan immunosuppressive drug. Non-limiting examples of immunosuppressivedrugs include but are not limited to a glucocorticoid, a cytostatic, anantibody, an immunophilin inhibitor, ciclosporin, tacrolimus, sirolimusand interferon, an opioid, a TNF binding protein, a cyclooxygenaseinhibitor, an antihistamine, an antimetabolite, folic acid,methotrexate, a purine analogue, a pyrimidine analogue, a proteinsynthesis inhibitor, mycophenolate, a cytotoxic antibiotic, a steroid,an anti-TNF antibody, a TNF inhibitor, and an NSAID. Exosomecompositions comprise anti-TNF antibodies, including but not limited toinfliximab, adalimumab, certolizumab, and golimumab. Exosomecompositions comprise TNF inhibitors including but not limited toetanercept, xanthine derivatives, and bupropion. In some cases, exosomecompositions comprise one or more NSAIDs. In some cases, exosomecompositions comprise at least one steroid.

Exosome compositions, methods of isolation, and methods and uses intreating disease are illustrated by the drawings provided herein. FIGS.1A-1E show identification and isolation of novel CD39+CD362+ stromalcell population from human umbilical cord. FIG. 1A shows arepresentative gating strategy. FIG. 1B shows identification ofCD39+CD362+(highlighted in bolded square gate and arrow) stromal cellpopulation. FIG. 1C shows CD362+0.121±0.054, CD39+CD362+0.105±0.045(n=5% mean±SEM). FIG. 1D and FIG. 1E show CFU-f analysis and example ofcolonies formed from CD39+CD362+ sorted (FIG. 1D) and wildtype (FIG. 1E,WT unsorted) populations by Miltenyi MACSQuant Tyto microchip cellsorter, n=2.

FIGS. 2A-C show identification and isolation of novel CD39+CD362+stromal cell population from human bone marrow. FIG. 2A shows arepresentative gating controls and identification ofCD39+CD362+(highlighted in bolded square gate) stromal cell populations.FIG. 2B shows CD362+0.4093±0.103, CD39+CD362+0.0147±0.002, (n=3%mean±SEM). FIG. 2C shows CFU-f analysis of colonies formed from wildtype(WT unsorted), CD362+, CD39+CD362+, CD39+ and double negative (−/−)populations by BD FACSAria cell sorter, n=3.

FIGS. 3A-B show identification and isolation of novel CD271+CD362+stromal cell population from human bone marrow using a novel closedmicrochip cell sorter Miltenyi Tyto. FIG. 3A shows a representativeMiltenyi MACSQuant Tyto sort gate and post sort analysis gating strategyfor identification of CD39+CD362+(highlighted in bolded square gate)stromal cell population, n=1. FIG. 3B shows CFU-f analysis and exampleof colonies formed from CD39+CD362+ populations isolated by BD FACSAriacell sorter (n=3) and Miltenyi MACSQuant Tyto microchip cell sorter,(n=1).

FIG. 4 provides a cartoon depiction of an exosome as contemplatedherein. In this drawing, a number of components important to someexosomes are shown including adhesion molecules such as ICAMs andintegrins; tetraspanins such as CD63, CD9, and CD81; cytoskeletonproteins such as actin, tubulin ERMs, and myosin; membrane traffickingproteins such as RABs and annexins; endosomal molecules such as TSG101,ubiquitin, clatherin, and ALIX; and lipid rafts including ceramide,cholesterol, and phosphatidylserine; nucleic acids such as mRNAmolecules, for example mRNA molecules encoding a protein or proteins ofinterest, DNA fragments or entire DNA coding molecules of variouslengths up to and including DNA molecules harboring multiple codingloci, miRNA or pre-miRNA, such as miRNA or pre-miRNA impacting heexpression of a gene or transcript encoding a protein involved in animmune response. None, any or all of these components, up to andincluding various permutations or combinations up to all of thesecomponents or variants of one or more of these components, are presentin some various SDC2+ exosomes as contemplated herein.

FIG. 5 shows SDC2 presence in exosomes derived from SDC2+ mesenchymalstromal cells (MSC) by transmission electron microscopy to detectgold-tagged anti-SDC2 antibody binding. Expression of CD63, TSG101 andAlix is similarly shown by transmission electron microscopy ofmetal-labeled antibodies to the respective proteins. The scale bar ineach photomicrograph is 100 nm. The figure demonstrates that SDC2, CD63,TSG101 and Alix are present in the exosomes isolated from SDC2+mesenchymal stem cells. The isotype control demonstrates that theantibodies bind specifically. The lack of Exosome labeling with a ratIgG2B APC-conjugated Isotype Control antibody (Cat #IC013A from R&DSystems) indicates the specificity of the anti-SDC2 labeling with anequivalent rat IgG2B APC-conjugated anti-SDC2 antibody.

FIG. 6 shows SDC2 expression in exosomes derived from SDC2+ mesenchymalstromal cells (MSC) by protein gel electrophoresis. In this experiment,H36 (marrow derived SDC2+MSC), H37 (marrow derived SDC2+ MSC), and HUC12(human umbilical cord derived SDC2+ MSC) cells and exosomes purifiedfrom those cells were homogenized in lysis buffer and run on an SDS-PAGEgel, along with unpurified whole sample protein extracts. Proteins fromthe gel were transferred to a membrane and stained with a rat IgG2Banti-human SDC2 antibody. In each ‘exo’ exosome protein extract lane,strong enrichment at below 50 kDa is observed, indicative of SDC2/CD362being disproportionately present in the exosome fraction relative to thewhole cell extract fraction.

SDC2+ exosomes were tested for their safety and efficacy to reduceinflammation-related damage in a mammalian model of inflammation damage.

FIG. 7 shows arterial FI 0.3 in rats treated with phosphate bufferedsaline (‘PBS’) vehicle, human umbilical cord derived SDC2+ stromalcells, or exosomes in a rat model of acute respiratory distress syndrome(‘ARDS’). In this experiment, 300 g Sprague Dawley rats were given anintrapulmonary dose of E. coli to induce ARDS lung injury. One hourlater, the rats were treated with a 90-100 mg dose of exosomes or 4million human mesenchymal stem cells (hMSC). The extent of lung injurywas then measured 24 hours later by measurement of arterial oxygen at FI0.3. This experiment shows that rats treated with only 90-100 μg ofexosomes had improved lung function relative to those treated with PBSvehicle. It is expected that rats treated with a greater dose showincreased improvement in lung function, such as at a level comparable tothat of hMSC treatment.

FIG. 8 shows arterial FI 1.0 in rats treated with PBS vehicle, humanumbilical cord derived SDC2+ stromal cells, or exosomes in a rat modelof ARDS. In this experiment, 300 g Sprague Dawley rats were given anintrapulmonary dose of E. coli to induce ARDS lung injury. One hourlater, the rats were treated with a 90-100 mg dose of exosomes or 4million human umbilical cord derived SDC2+ stromal cells. The extent oflung injury was then measured 24 hours later by measurement of arterialoxygen at FI 1.0. This experiment shows that rats treated with only90-100 mg of exosomes had no decrease in lung function than thosetreated with PBS vehicle. This result indicates that exosomeadministration is not deleterious to animal lung recovery.

FIG. 9 shows arterial FI 0.3 in rats treated with PBS vehicle, bonemarrow (BM) exosomes, or human umbilical cord (HUC) exosomes in a ratmodel of ARDS. In this experiment, 300 g Sprague Dawley rats were givenan intrapulmonary dose of E. coli to induce ARDS lung injury, one hourlater, the rats were treated with a 90-100 mg dose of exosomes purifiedfrom bone marrow mesenchymal stem cells or human umbilical cordmesenchymal stem cells. The extent of lung injury was then measured 24hours later by measurement of arterial oxygen at FI 0.3. This experimentshows that rats treated with only 90-100 mg of exosomes had no decreasein lung function than those treated with PBS vehicle. It is expectedthat rats treated with a greater dose greater dose show increasedimprovement in lung function.

FIG. 10 shows arterial FI 1.0 in rats treated with PBS vehicle, bonemarrow (BM) exosomes, or human umbilical cord (HUC) exosomes in a ratmodel of ARDS. In this experiment, 300 g Sprague Dawley rats were givenan intrapulmonary dose of E. coli to induce ARDS lung injury, one hourlater, the rats were treated with a 90-100 mg dose of exosomes purifiedfrom bone marrow mesenchymal stem cells or human umbilical cordmesenchymal stem cells. The extent of lung injury was then measured 24hours later by measurement of arterial oxygen at FI 1.0. This experimentshows that rats treated with only 90-100 μg of exosomes had no decreasein lung function than those treated with PBS vehicle. It is expectedthat rats treated with a greater dose show increased improvement in lungfunction.

FIG. 11 shows bronchioaveolar lavage (BAL) bacterial load in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated with a 90-100 μg dose of exosomes or 4million human mesenchymal stem cells (hMSC). The extent of lung injurywas then measured 24 hours later by measurement of BAL bacterial load.This experiment shows that rats treated with only 90-100 μg of exosomeshad reduced BAL bacterial load than those treated with PBS vehicle,demonstrating the efficacy of administering exosomes as a treatment. Itis expected that rats treated with a greater dose show increasedimprovement in lung function.

FIG. 12 shows bronchioaveolar lavage (BAL) total cell count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated with a 90-100 μg dose of exosomes or 4million human mesenchymal stem cells (hMSC). The extent of lung injurywas then measured 24 hours later by measurement of BAL total cell count.This experiment shows that rats treated with only 90-100 μg of exosomeshad reduced BAL total cell count compared to those treated with PBSvehicle, demonstrating the efficacy of administering exosomes as atreatment. It is expected that rats treated with a greater dose showeven better improvement in lung function.

FIG. 13 shows bronchioaveolar lavage (BAL) neutrophil count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated with a 90-100 μg dose of exosomes or 4million human mesenchymal stem cells (hMSC). The extent of lung injurywas then measured 24 hours later by measurement of BAL neutrophil count.This experiment shows that rats treated with only 90-100 μg of exosomeshad fewer infiltrating neutrophils in the lung than those treated withPBS vehicle, demonstrating the efficacy of administering exosomes as atreatment. It is expected that rats treated with a greater dose showeven better improvement in lung function.

FIG. 14 shows static lung compliance in rats treated with PBS vehicle,human mesenchymal stem cells, or exosomes in a rat model of ARDS. Inthis experiment, 300 g Sprague Dawley rats were given an intrapulmonarydose of E. coli to induce ARDS lung injury, one hour later, the ratswere treated with a 90-100 μg dose of exosomes or 4 million humanmesenchymal stem cells (hMSC). The extent of lung injury was thenmeasured 24 hours later by measurement of static lung compliance. Thisexperiment shows that rats treated with only 90-100 μg of exosomes hadno decrease in lung function than those treated with PBS vehicle. Thisresult indicates that exosome administration is not deleterious toanimal lung recovery.

FIG. 15 shows lung wet dry ratio in rats treated with PBS vehicle, humanmesenchymal stem cells, or exosomes in a rat model of ARDS. In thisexperiment, 300 g Sprague Dawley rats were given an intrapulmonary doseof E. coli to induce ARDS lung injury, one hour later, the rats weretreated with a 90-100 μg dose of exosomes or 4 million human mesenchymalstem cells (hMSC). The extent of lung injury was then measured 24 hourslater by measurement of lung wet dry ratio. This experiment shows thatrats treated with only 90-100 μg of exosomes had no decrease in lungfunction than those treated with PBS vehicle. This result indicates thatexosome administration is not deleterious to animal lung recovery.

FIG. 16 shows results from a scratch assay with A549 cells treated withserum free media or exosomes for 48 hours. The scratch assay measurescell migration into a scratch created in adherent cells growing in aculture dish. In this assay, 300,000 A549 cells were grown in amonolayer in a 24 well plate. The cells were scraped with a p200 pipettip in a straight line to create a scratch. An image was taken at thetime of scraping (0 h) and after a 48 hour incubation with serum freemedia or 2 μg exosomes in serum free media. Exosomes are shown there toreduce the size of the scratch, therefore increasing cell migration ofA549 cells compared to the serum free media control. These resultsindicate that exosomes increase epithelial cell migration.

FIG. 17 shows results a scratch assay with A549 cells, plate 1. An imagewas taken at the time of scraping (0 h) and after a 48 hour incubationwith serum free media or 2 μg exosomes in serum free media. Exosomes areshown there to reduce the size of the scratch, therefore increasing cellmigration of A549 cells compared to the serum free media control. Theseresults indicate that exosomes increase epithelial cell migration.

FIG. 18 shows a bar graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultrafiltration. In this assay, 30,000 A549 cells that weretransfected with an NFκB-luciferase reporter gene were grown in a 96well dish. The cells were treated with serum free media or 2 μg exosomesin serum free media for hours and then stimulated with human IL-1β forhours and luciferase activity was measured. A reduction was observed inluciferase activity in A549 cells treated with exosomes. Therefore,SDC2+ exosomes reduce NFκB activation.

FIG. 19 shows a scatter graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultrafiltration. In this assay, 30,000 A549 cells that weretransfected with an NFκB-luciferase reporter gene were grown in a 96well dish. The cells were treated with serum free media or 2 μg exosomesin serum free media and then stimulated with human IL-1β for hours andluciferase activity was measured. A reduction was observed in luciferaseactivity in A549 cells treated with exosomes. These results indicatethat the exosome administration reduces NFκB activation.

FIG. 20 shows a bar graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultracentrifugation. In this assay, 30,000 A549 cells that weretransfected with an NFκB-luciferase reporter gene were grown in a 96well dish. The cells were treated with serum free media or 2 μg exosomesin serum free media for hours and then stimulated with human IL-1β forhours and luciferase activity was measured. A reduction was observed inluciferase activity in A549 cells treated with exosomes. Therefore,exosomes reduce NFκB activation.

FIG. 21 shows a scatter graph of NFκB reporter gene activation with IL1βstimulation in cells treated with serum free media or exosomes purifiedby ultracentrifugation. In this assay, 30,000 A549 cells that weretransfected with an NFκB-luciferase reporter gene were grown in a 96well dish. The cells were treated with serum free media or 2 μg exosomesin serum free media for hours and then stimulated with human IL-1β forhours and luciferase activity was measured. A reduction was observed inluciferase activity in A549 cells treated with exosomes. These resultsindicate that the exosome administration reduces NFκB activation.

FIG. 22 shows arterial FI 0.3 in rats treated with PBS vehicle, hMSC, orexosomes in a rat model of ARDS. In this experiment, 300 g SpragueDawley rats were given an intrapulmonary dose of E. coli to induce ARDSlung injury, one hour later, the rats were treated with a 200 μg dose ofexosomes or 10 million/kg human MSC by intravenous administration. Theextent of lung injury was then measured 24 hours later by measurement ofarterial oxygen at FI 0.3. This experiment shows that rats treated with200 μg of exosomes or 10 million/kg human MSC had significantly improvedlung function compared to those treated with PBS vehicle (one way anovap<0.0001). This result indicates that exosome administration iseffective in treating animal lung injury and suggests that exosometreatment is comparable to hMSC treatment.

FIG. 23 shows arterial FI 1.0 in rats treated with PBS vehicle, hMSC, orexosomes in a rat model of ARDS. In this experiment, 300 g SpragueDawley rats were given an intrapulmonary dose of E. coli to induce ARDSlung injury, one hour later, the rats were treated with a 200 μg dose ofexosomes or 10 million/kg human MSC by intravenous administration. Theextent of lung injury was then measured 24 hours later by measurement ofarterial oxygen at FI 0.3. This experiment shows that rats treated with200 μg of exosomes or 10 million/kg human MSC had significantly improvedlung function compared to those treated with PBS vehicle (one way anovap<0.0001). This result indicates that exosome administration iseffective in treating animal lung injury and suggests that exosometreatment is comparable to hMSC treatment.

FIG. 24 shows static lung compliance in rats treated with PBS vehicle,human mesenchymal stem cells, or exosomes in a rat model of ARDS. Inthis experiment, 300 g Sprague Dawley rats were given an intrapulmonarydose of E. coli to induce ARDS lung injury, one hour later, the ratswere treated with a 200 mg intravenous dose of exosomes or 10 million/kghuman mesenchymal stem cells (hMSC). The extent of lung injury was thenmeasured 24 hours later by measurement of static lung compliance. Thisexperiment shows that rats treated with 200 mg of exosomes or 10million/kg human MSC had significantly improved lung function comparedto those treated with PBS vehicle (one way anova p<0.01). This resultindicates that exosome administration is effective in treating animallung injury and suggests that exosome treatment is comparable to hMSCtreatment.

FIG. 25 shows bronchioaveolar lavage (BAL) bacterial load in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated intravenously with a 200 mg dose ofexosomes or 10 million/kg human mesenchymal stem cells (hMSC). Theextent of lung injury was then measured 24 hours later by measurement ofBAL bacterial load. This experiment shows that rats treated withexosomes or hMSC had a significantly reduced BAL bacterial load thanthose treated with PBS vehicle (one way anova p<0.01), demonstrating theefficacy of administering exosomes as a treatment and suggests thatexosome treatment is comparable to hMSC treatment.

FIG. 26 shows bronchioaveolar lavage (BAL) total cell count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated intravenously with a 200 mg dose ofexosomes or 10 million/kg human mesenchymal stem cells (hMSC). Theextent of lung injury was then measured 24 hours later by measurement ofBAL total cell count. This experiment shows that rats treated withexosomes or hMSC had a significantly reduced BAL total cell countcompared to those treated with PBS vehicle (one way anova p<0.01),demonstrating the efficacy of administering exosomes as a treatment andsuggests that exosome treatment is comparable to hMSC treatment.

FIG. 27 shows bronchioaveolar lavage (BAL) neutrophil count in ratstreated with PBS vehicle, human mesenchymal stem cells, or exosomes in arat model of ARDS. In this experiment, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated intravenously with a 200 mg dose ofexosomes or 10 million/kg human mesenchymal stem cells (hMSC). Theextent of lung injury was then measured 24 hours later by measurement ofBAL neutrophil count. This experiment shows that rats treated withexosomes or hMSC had significantly reduced infiltrating neutrophils inthe lung than those treated with PBS vehicle, demonstrating the efficacyof administering exosomes as a treatment and suggests that exosometreatment is comparable to hMSC treatment. Throughout the figures, asignificant difference is indicated by an asterisk (*).

The disclosure herein is further clarified in reference to a partiallist of numbered embodiments as follows. 1. A method of isolating apopulation of SDC2+ stromal stem cells from a mixed population ofmammalian cells the method comprising: (a) contacting the mixedpopulation of mammalian cells to a CD39 binding agent; (b) isolatingcells bound to the CD39 binding agent; and (c) measuring SDC2+ cellabundance in the isolated cells, thereby isolating the population ofSDC2+ stromal stem cells. 2. The method of embodiment 1, wherein thepopulation of SDC2+ stromal stem cells comprise cells selected from alist consisting of human, mouse, rat, and equine cells. 3. The method ofembodiment 1 or embodiment 2, wherein the mixed population of mammaliancells are obtained from a source selected from at least one of bonemarrow, adipose tissue, skeletal muscle, endometrium, placenta,umbilical cord, Wharton's jelly, and cells derived from pluripotentcells. 4. The method of any one of embodiments 1 to 3, wherein the CD39binding agent comprises an antibody. 5. The method of embodiment 4,wherein the antibody is raised to a CD39 antigen. 6. The method ofembodiment 4, wherein the antibody comprises a variable domain thatspecifically binds to at least one mammalian CD39 protein. 7. The methodof embodiment 4, wherein the antibody specifically binds to at least oneof a human, a mouse, a rat, and equine CD39 protein. 8. The method ofembodiment 4, wherein the antibody is conjugated to a fluorophore. 9.The method of embodiment 4, wherein the antibody is conjugated to abead. 10. The method of any one of embodiments 1 to 9, wherein at least20% of the population of SDC2+ stromal stem cells is SDC2+. 11. Themethod of any one of embodiments 1 to 10, wherein at least 40% of thepopulation of SDC2+ stromal stem cells is SDC2+. 12. The method of anyone of embodiments 1 to 11, wherein at least 70% of the population ofSDC2+ stromal stem cells is SDC2+. 13. The method of any one ofembodiments 1 to 12, wherein isolating the cells bound to the CD39binding agent comprises fluorescence activated cell sorting. 14. Themethod of any one of embodiments 1 to 12, wherein isolating the cellsbound to the CD39 binding agent comprises magnetic-activated cellsorting. 15. The method of any one of embodiments 1 to 14, comprisingculturing the isolated cells. 16. The method of any one of embodiments 1to 15, wherein at least 90% of the SDC2+ stromal stem cells are CD45−.17. The method of any one of embodiments 1 to 16, wherein the cellsbound to the CD39 binding agent further comprise CD25+ FoxP3+ regulatoryT cells. 18. The method of any one of embodiments 1 to 17, comprisinggenetically modifying the cells to overexpress an apyrase. 19. Themethod of any one of embodiments 1 to 18, comprising geneticallymodifying the cells to overexpress a protein having a CD39 extracellulardomain. 20. The method of any one of embodiments 1 to 19, comprisinggenetically modifying the cells to overexpress CD39. 21. The method ofany one of embodiments 1 to 20, comprising genetically modifying thecells to overexpress CD39L3. 22. A method of preparing animmunomodulatory composition comprising a population of SDC2+ stromalstem cells, the method comprising (a) contacting a mixed population ofmammalian cells to a CD39 binding agent; (b) isolating cells bound tothe CD39 binding agent; and (c) measuring SDC2+ cell abundance in theisolated cells. 23. The method of embodiment 22, wherein the populationof SDC2+ stromal stem cells is a population of human, a mouse, a rat, oran equine cells. 24. The method of embodiment 22 or embodiment 23,wherein the mixed population of mammalian cells are obtained from asource selected from at least one of bone marrow, adipose tissue,skeletal muscle, endometrium, placenta, umbilical cord, Wharton's jelly,and cells derived from pluripotent cells. 25. The method of any one ofembodiments 22 to 24, wherein the CD39 binding agent comprises anantibody. 26. The method of embodiment 25, wherein the antibody israised to a CD39 antigen. 27. The method of embodiment 25, wherein theantibody comprises a variable domain that specifically binds to at leastone mammalian CD39 protein. 28. The method of embodiment 25, wherein theantibody specifically binds to at least one of a human, a mouse, a rat,and equine CD39 protein. 29. The method of embodiment 25, wherein theantibody is conjugated to a fluorophore. 30. The method of embodiment25, wherein the antibody is conjugated to a bead. 31. The method of anyone of embodiments 22 to 30, wherein at least 20% of the population ofSDC2+ stromal stem cells is SDC2+. 32. The method of any one ofembodiments 22 to 31, wherein at least 40% of the population of SDC2+stromal stem cells is SDC2+. 33. The method of any one of embodiments 22to 32, wherein at least 70% of the population of SDC2+ stromal stemcells is SDC2+. 34. The method of any one of embodiments 22 to 33,wherein isolating the cells bound to the CD39 binding agent comprisesfluorescence activated cell sorting. 35. The method of any one ofembodiments 22 to 33, wherein isolating the cells bound to the CD39binding agent comprises magnetic-activated cell sorting. 36. The methodof any one of embodiments 22 to 35, comprising culturing the cellsisolated cells. 37. The method of any one of embodiments 22 to 36,wherein at least 90% of the SDC2+ stromal stem cells are CD45−. 38. Themethod of any one of embodiments 22 to 37, wherein the compositionfurther comprises a buffer. 39. The method of any one of embodiments 22to 38, wherein the composition further comprises an extracellularmatrix. 40. The method of embodiment 39, wherein the extracellularmatrix is comprises a collagen. 41. The method of embodiment 39, whereinthe extracellular matrix is comprises a hyaluronic acid. 42. The methodof any one of embodiments 22 to 41, wherein the composition furthercomprises a cryopreservant comprising DMSO. 43. The method of any one ofembodiments 22 to 41, wherein the composition further comprises acryopreservant comprising glycerol. 44. The method of any one ofembodiments 22 to 43, wherein the population of SDC2+ stromal stem cellsfurther comprises CD25+ FoxP3+ regulatory T cells. 45. The method of anyone of embodiments 22 to 44, comprising genetically modifying the cellsto overexpress an apyrase. 46. The method of any one of embodiments 22to 45, comprising genetically modifying the cells to overexpress CD39.47. The method of any one of embodiments 22 to 46, comprisinggenetically modifying the cells to overexpress CD39L3. 48. A method ofisolating an immuno-modulatory composition comprising exosomes, themethod comprising: (a) contacting a population of mammalian cells to aCD39 binding agent; (b) isolating the cells bound to the CD39 bindingagent; and (c) recovering a supernatant comprising the exosomes from theisolated cells. 49. The method of embodiment 48, comprising obtaining anexosome fraction from the supernatant. 50. The method of embodiment 49,wherein obtaining an exosome fraction comprises centrifuging thesupernatant. 51. The method of embodiment 50, wherein the centrifugationcomprises centrifuging the cells at about 100,000 g. 52. The method ofembodiment 50 or embodiment 51, wherein the centrifugation comprisescentrifuging the cells for at least one hour. 53. The method of any oneof embodiments 50 to 52, wherein the centrifugation comprisesultrafiltration. 54. The method of any one of embodiments 50 to 53,wherein the centrifugation comprises size-exclusion liquidchromatography. 55. The method of any one of embodiments 49 to 54,wherein obtaining an exosome fraction comprises ultrafiltration. 56. Themethod of any one of embodiments 49 to 55, wherein obtaining an exosomefraction comprises size-exclusion liquid chromatography. 57. The methodof any one of embodiments 49 to 56, wherein obtaining an exosomefraction comprises contacting the supernatant to an antibody. 58. Themethod of embodiment 57, wherein the antibody is selected from at leastone of an anti-CD39 antibody and an anti-SDC2 antibody. 59. The methodof any one of embodiments 48 to 58, wherein the exosomes are paracrinesignaling exosomes. 60. The method of any one of embodiments 48 to 59,wherein the isolated cells are SDC2+. 61. The method of any one ofembodiments 48 to 60, wherein the isolated cells comprise mesenchymalstem cells. 62. The method of any one of embodiments 48 to 61, whereinat least 90% of the isolated cells are CD45−. 63. The method of any oneof embodiments 48 to 62, wherein the method comprises storing theexosome fraction at room temperature. 64. The method of any one ofembodiments 48 to 63, wherein the method comprises storing the exosomefraction without cryogenic preservation. 65. The method of any one ofembodiments 48 to 64, wherein the method comprises adding animmunosuppressive drug to the immuno-modulatory composition. 66. Themethod of any one of embodiments 48 to 65, wherein the isolated cellsare perturbed to elicit exosome production. 67. The method of any one ofembodiments 48 to 66, wherein the isolated cells are cultured in ahollow-fiber bioreactor. 68. The method of any one of embodiments 48 to67, wherein the isolated cells further comprise CD25+ FoxP3+ regulatoryT cells. 69. The method of any one of embodiments 48 to 68, comprisinggenetically modifying the isolated cells to overexpress an apyrase. 70.The method of any one of embodiments 48 to 69, comprising geneticallymodifying the isolated cells to overexpress CD39. 71. The method of anyone of embodiments 48 to 70, comprising genetically modifying theisolated cells to overexpress CD39L3. 72. A method of modulating aninflammation response in a mammal comprising delivering a compositioncomprising SDC2+ cells to a site of the inflammation response, whereinthe SDC2+ cells are isolated from a mixed population of cells byisolating cells based upon CD39 expression. 73. The method of embodiment72, wherein isolating cells comprises collecting cells bound to a CD39binding agent. 74. The method of embodiment 73, wherein isolating thecells comprises culturing the cells bound to the CD39 binding agent. 75.The method of any one of embodiments 72 to 74, wherein deliveringcomprises injecting the composition comprising SDC2+ cells. 76. Themethod of any one of embodiments 72 to 74, wherein delivering comprisestopically applying the composition comprising SDC2+ cells. 77. Themethod of embodiment 76, wherein the composition comprises a hydrogel.78. The method of embodiment 76, wherein the composition comprises acollagen gel. 79. The method of any one of embodiments 72 to 74, whereindelivering comprises intraocularly administering the compositioncomprising SDC2+ cells. 80. The method of any one of embodiments 72 to74, wherein delivering comprises ophthalmic application of thecomposition comprising SDC2+ cells. 81. The method of any one ofembodiments 72 to 74, wherein delivering comprises intravenous deliverythe composition comprising SDC2+ cells. 82. The method of any one ofembodiments 72 to 74, wherein delivering comprises intra-lymph nodeinjection of the composition comprising SDC2+ cells. 83. The method ofany one of embodiments 72 to 74, wherein delivering comprisessubcutaneous delivery of the composition comprising SDC2+ cells. 84. Themethod of any one of embodiments 72 to 74, wherein delivering comprisesintraperitoneal delivery of the composition comprising SDC2+ cells. 85.The method of any one of embodiments 72 to 74, wherein deliveringcomprises intrathecal delivery of the composition comprising SDC2+cells. 86. The method of any one of embodiments 72 to 85, wherein theSDC2+ cells wherein the SDC2+ cells are selected from at least one ofhuman, mouse, rat, and equine cells. 87. The method of any one ofembodiments 72 to 86, wherein the mixed population of mammalian cellsare obtained from a source selected from bone marrow, adipose tissue,skeletal muscle, endometrium, placenta, umbilical cord, Wharton's jelly,and cells derived from pluripotent cells. 88. The method of any one ofembodiments 72 to 87, wherein the CD39 binding agent comprises anantibody. 89. The method of embodiment 88, wherein the antibody israised to a CD39 antigen. 90. The method of embodiment 88, wherein theantibody comprises a variable domain that specifically binds to at leastone mammalian CD39 protein. 91. The method of embodiment 88, wherein theantibody specifically binds to at least one of a human, a mouse, a rat,or equine CD39 protein. 92. The method of embodiment 88, wherein theantibody is conjugated to a fluorophore. 93. The method of embodiment88, wherein the antibody is conjugated to a bead. 94. The method of anyone of embodiments 72 to 93, wherein at least 20% of the SDC2+ cells areSDC2+. 95. The method of any one of embodiments 72 to 94, wherein atleast 40% of the SDC2+ cells are SDC2+. 96. The method of any one ofembodiments 72 to 95, wherein at least 70% of the SDC2+ cells are SDC2+.97. The method of any one of embodiments 72 to 96, wherein isolating thecells comprises fluorescence activated cell sorting. 98. The method ofany one of embodiments 72 to 96, wherein isolating the cells comprisesmagnetic-activated cell sorting. 99. The method of any one ofembodiments 72 to 98, wherein at least 90% of the SDC2+ cells are CD45−.100. The method of any one of embodiments 72 to 99, wherein the methodcomprises delivering at least 10{circumflex over ( )}3 SDC2+ cells. 101.The method of any one of embodiments 72 to 100, wherein the methodcomprises delivering at least 10{circumflex over ( )}4 SDC2+ cells. 102.The method of any one of embodiments 72 to 101, wherein the methodcomprises delivering at least 10{circumflex over ( )}5 SDC2+ cells. 103.The method of any one of embodiments 72 to 102, wherein the methodcomprises delivering at least 10{circumflex over ( )}6 SDC2+ cells. 104.The method of any one of embodiments 72 to 103, wherein the isolatedcells comprise CD25+ FoxP3+ regulatory T cells. 105. The method of anyone of embodiments 72 to 104, comprising genetically modifying theisolated cells to overexpress an apyrase. 106. The method of any one ofembodiments 72 to 105, comprising genetically modifying the isolatedcells to overexpress CD39. 107. The method of any one of embodiments 72to 106, comprising genetically modifying the isolated cells tooverexpress CD39L3. 108. The method of any one of embodiments 72 to 107,wherein the inflammation response comprises at least one of type 1diabetes, type 2 diabetes, sepsis, Crohn's disease, inflammatory bowelsyndrome, rheumatoid arthritis, graft versus host disease, multiplesclerosis, ALS, a dermal wound, a bone fracture, a concussion wound, aburn, atherosclerosis, nephropathy, cardiomyopathy, neuropathy, a kidneydisorder, kidney failure, a diabetic ulcer, a leg ulcer, ARDS, sepsis,inflammatory liver disease, myocarditis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, subacute bacterial endocarditis,anti-glomulerular basement membrane nephritis, interstitial cystitis,lupus enphritis, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, antisynthetase syndrome, asthma, chronicobstructive pulmonary disease, cystic fibrosis, atelectasis, bronchitis,emphasema, pneumonia, pulmonary endema, Alopecia Areata, autoimmuneAngioedema, autoimmune progesterone dermatitis, autoimmune urticaria,Bullous pemphigoid, Cicatricial pemphigoid, Dermatitis herpetiformis,Discoid lupus erythematosus, Epidermolysis bullosa acquisita, Erythemanodosum, Gestational pemphigoid, Hidradenitis suppurativa, Lichenplanus, Lichen sclerosus, Linear IgA disease, Morphea, Pemphigusvulgaris, Pityriasis lichenoides et varioliformis acuta, Mucha-Habermanndisease, Psoriasis, Systemic scleroderma, Vitiligo, Addison's disease,autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrometype 2, autoimmune polyendocrine syndrome type 3, autoimmunepancreatitis, autoimmune thyroiditis, Ord's thyroiditis, Graves'disease, Reproductive Organ disorder, autoimmune oophoritis,Endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmuneenteropathy, Celiac disease, Microscopic colitis, Ulcerative colitis,Antiphospholipid syndrome, Aplastic anemia, autoimmune hemolytic anemia,autoimmune lymphoproliferative syndrome, autoimmune neutropenia,autoimmune thrombocytopenic purpura, Cold agglutinin disease, Essentialmixed cryoglobulinemia, Evans syndrome, IgG4-related systemic disease,Paroxysmal nocturnal hemoglobinuria, Pernicious anemia, Pure red cellaplasia, Thrombocytopenia, Adiposis dolorosa, Adult-onset Still'sdisease, Ankylosing Spondylitis, CREST syndrome, Drug-induced lupus,Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome,Juvenile Arthritis, Lyme disease (Chronic), Mixed connective tissuedisease, Palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis,Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic LupusErythematosus, Undifferentiated connective tissue disease, cachexia,sarcophenia, Dermatomyositis, Fibromyalgia, Inclusion body myositis,Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellardegeneration, Polymyositis, Acute disseminated encephalomyelitis, Acutemotor axonal neuropathy, Anti-N-Methyl-D-Aspartate ReceptorEncephalitis, Balo concentric sclerosis, Bickerstaffs encephalitis,Chronic inflammatory demyelinating polyneuropathy, Guillain-Barrésyndrome, Hashimoto's encephalopathy, Idiopathic inflammatorydemyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiplesclerosis, Pediatric Autoimmune Neuropsychiatric Disorder Associatedwith Streptococcus, Progressive inflammatory neuropathy, Restless legsyndrome, Stiff person syndrome, Sydenham chorea, amyotrophic lateralsclerosis, Parkinson's disease, Alzheimer's disease, Transversemyelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome,Graves ophthalopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, autoimmune inner ear disease, Meniere's disease,Anti-neutrophil cytoplasmic antibody-associated vasculitis, Behçet'sdisease, Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonleinpurpura, Kawasaki's disease, Leukocytoclastic vasculitis, Lupusvasculitis, Rheumatoid vasculitis, Microscopic polyangiitis,Polyarteritis nodosa, Polymyalgia rheumatica, Urticarial vasculitis, andVasculitis. 109. A method of modulating an inflammation response in amammal comprising delivering a composition comprising exosomes to a siteof the inflammation response, wherein the exosomes are SDC2+ and theexosomes are from SDC2+ cells isolated from a mixed population ofmammalian cells based upon CD39 expression. 110. The method ofembodiment 109, wherein isolating cells comprises collecting cells boundto a CD39 binding agent. 111. The method of embodiment 109 or embodiment110, wherein isolating the cells comprises culturing the cells bound tothe CD39 binding agent. 112. The method of any one of embodiments 109 to111, wherein delivering comprises injecting the composition comprisingexosomes. 113. The method of any one of embodiments 109 to 111, whereindelivering comprises topically applying the composition comprisingexosomes. 114. The method of embodiment 113, wherein the compositioncomprises a hydrogel. 115. The method of embodiment 113, wherein thecomposition comprises a collagen gel. 116. The method of any one ofembodiments 109 to 111, wherein delivering comprises intraocularlyadministering the composition comprising exosomes. 117. The method ofany one of embodiments 109 to 111, wherein delivering comprisesophthalmic application of the composition comprising exosomes. 118. Themethod of any one of embodiments 109 to 111, wherein deliveringcomprises intravenous delivery the composition comprising exosomes. 119.The method of any one of embodiments 109 to 111, wherein deliveringcomprises intra-lymph node injection of the composition comprisingexosomes. 120. The method of any one of embodiments 109 to 111, whereindelivering comprises subcutaneous delivery of the composition comprisingexosomes. 121. The method of any one of embodiments 109 to 111, whereindelivering comprises intraperitoneal delivery of the compositioncomprising exosomes. 122. The method of any one of embodiments 109 to111, wherein delivering comprises intrathecal delivery of thecomposition comprising exosomes. 123. The method of any one ofembodiments 109 to 122, wherein the exosomes are isolated from SDC2+cells that are at least one of human, mouse, rat, and equine cells. 124.The method of any one of embodiments 109 to 123, wherein the mixedpopulation of mammalian cells are obtained from a source selected frombone marrow, adipose tissue, skeletal muscle, endometrium, placenta,umbilical cord, Wharton's jelly, and cells derived from pluripotentcells. 125. The method of any one of embodiments 109 to 124, wherein theCD39 binding agent comprises an antibody. 126. The method of embodiment125, wherein the antibody is raised to a CD39 antigen. 127. The methodof embodiment 125, wherein the antibody comprises a variable domain thatspecifically binds to at least one mammalian CD39 protein. 128. Themethod of embodiment 125, wherein the antibody specifically binds to atleast one of a human, a mouse, a rat, or equine CD39 protein. 129. Themethod of embodiment 125, wherein the antibody is conjugated to afluorophore. 130. The method of embodiment 125, wherein the antibody isconjugated to a bead. 131. The method of any one of embodiments 109 to130, wherein at least 20% of the exosomes are SDC2+. 132. The method ofany one of embodiments 109 to 131, wherein at least 40% of the exosomesare SDC2+. 133. The method of any one of embodiments 109 to 132, whereinat least 70% of the exosomes are SDC2+. 134. The method of any one ofembodiments 109 to 133, wherein isolating the cells comprisesfluorescence activated cell sorting. 135. The method of any one ofembodiments 109 to 133, wherein isolating the cells comprisesmagnetic-activated cell sorting. 136. The method of any one ofembodiments 109 to 135, wherein the method further comprises culturingthe cells bound to the CD39 binding agent. 137. The method of any one ofembodiments 109 to 136, wherein the cells are CD45−. 138. The method ofany one of embodiments 109 to 137, wherein the method comprisesdelivering at least 10{circumflex over ( )}6 exosomes. 139. The methodof any one of embodiments 109 to 138, wherein the method comprisesdelivering at least 10{circumflex over ( )}7 exosomes. 140. The methodof any one of embodiments 109 to 139, wherein the method comprisesdelivering at least 10{circumflex over ( )}8 exosomes. 141. The methodof any one of embodiments 109 to 140, wherein the method comprisesdelivering at least 10{circumflex over ( )}9 exosomes. 142. The methodof any one of embodiments 109 to 141, wherein the isolated cellscomprise CD25+ FoxP3+ regulatory T cells. 143. The method of any one ofembodiments 109 to 142, comprising genetically modifying the isolatedcells to overexpress an apyrase. 144. The method of any one ofembodiments 109 to 143, comprising genetically modifying the isolatedcells to overexpress CD39. 145. The method of any one of embodiments 109to 144, comprising genetically modifying the isolated cells tooverexpress CD39L3. 146. The method of any one of embodiments 109 to145, wherein the inflammation response comprises at least one of type 1diabetes, type 2 diabetes, sepsis, Crohn's disease, inflammatory bowelsyndrome, rheumatoid arthritis, graft versus host disease, multiplesclerosis, ALS, a dermal wound, a bone fracture, a concussion wound, aburn, atherosclerosis, nephropathy, cardiomyopathy, neuropathy, a kidneydisorder, kidney failure, a diabetic ulcer, a leg ulcer, ARDS, sepsis,inflammatory liver disease, myocarditis, postmyocardial infarctionsyndrome, postpericardiotomy syndrome, subacute bacterial endocarditis,anti-glomulerular basement membrane nephritis, interstitial cystitis,lupus enphritis, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, antisynthetase syndrome, asthma, chronicobstructive pulmonary disease, cystic fibrosis, atelectasis, bronchitis,emphasema, pneumonia, pulmonary endema, Alopecia Areata, autoimmuneAngioedema, autoimmune progesterone dermatitis, autoimmune urticaria,Bullous pemphigoid, Cicatricial pemphigoid, Dermatitis herpetiformis,Discoid lupus erythematosus, Epidermolysis bullosa acquisita, Erythemanodosum, Gestational pemphigoid, Hidradenitis suppurativa, Lichenplanus, Lichen sclerosus, Linear IgA disease, Morphea, Pemphigusvulgaris, Pityriasis lichenoides et varioliformis acuta, Mucha-Habermanndisease, Psoriasis, Systemic scleroderma, Vitiligo, Addison's disease,autoimmune polyendocrine syndrome, autoimmune polyendocrine syndrometype 2, autoimmune polyendocrine syndrome type 3, autoimmunepancreatitis, autoimmune thyroiditis, Ord's thyroiditis, Graves'disease, Reproductive Organ disorder, autoimmune oophoritis,Endometriosis, autoimmune orchitis, Sjogren's syndrome, autoimmuneenteropathy, Celiac disease, Microscopic colitis, Ulcerative colitis,Antiphospholipid syndrome, Aplastic anemia, autoimmune hemolytic anemia,autoimmune lymphoproliferative syndrome, autoimmune neutropenia,autoimmune thrombocytopenic purpura, Cold agglutinin disease, Essentialmixed cryoglobulinemia, Evans syndrome, IgG4-related systemic disease,Paroxysmal nocturnal hemoglobinuria, Pernicious anemia, Pure red cellaplasia, Thrombocytopenia, Adiposis dolorosa, Adult-onset Still'sdisease, Ankylosing Spondylitis, CREST syndrome, Drug-induced lupus,Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome,Juvenile Arthritis, Lyme disease (Chronic), Mixed connective tissuedisease, Palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis,Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic LupusErythematosus, Undifferentiated connective tissue disease, cachexia,sarcophenia, Dermatomyositis, Fibromyalgia, Inclusion body myositis,Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellardegeneration, Polymyositis, Acute disseminated encephalomyelitis, Acutemotor axonal neuropathy, Anti-N-Methyl-D-Aspartate ReceptorEncephalitis, Balo concentric sclerosis, Bickerstaffs encephalitis,Chronic inflammatory demyelinating polyneuropathy, Guillain-Barrésyndrome, Hashimoto's encephalopathy, Idiopathic inflammatorydemyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiplesclerosis, Pediatric Autoimmune Neuropsychiatric Disorder Associatedwith Streptococcus, Progressive inflammatory neuropathy, Restless legsyndrome, Stiff person syndrome, Sydenham chorea, amyotrophic lateralsclerosis, Parkinson's disease, Alzheimer's disease, Transversemyelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome,Graves ophthalmopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, autoimmune inner ear disease, Meniere's disease,Anti-neutrophil cytoplasmic antibody-associated vasculitis, Behçet'sdisease, Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonleinpurpura, Kawasaki's disease, Leukocytoclastic vasculitis, Lupusvasculitis, Rheumatoid vasculitis, Microscopic polyangiitis,Polyarteritis nodosa, Polymyalgia rheumatica, Urticarial vasculitis, andVasculitis. 147. A composition comprising a population of cells that isat least 20% SDC2+, wherein the population of cells is bound to a CD39binding agent. 148. The composition of embodiment 147, wherein thepopulation of cells is at least 30% SDC2+. 149. The composition ofembodiment 147 or embodiment 148, wherein the population of cells is atleast 40% SDC2+. 150. The composition of any one of embodiments 147 to149, wherein the population of cells is at least 50% SDC2+. 151. Thecomposition of any one of embodiments 147 to 150, wherein the populationof cells is at least 60% SDC2+. 152. The composition of any one ofembodiments 147 to 151, wherein the population of cells is at least 70%SDC2+. 153. The composition of any one of embodiments 147 to 152,wherein the population of cells is at least 80% SDC2+. 154. Thecomposition of any one of embodiments 147 to 153, wherein the populationof cells is at least 90% SDC2+. 155. The composition of any one ofembodiments 147 to 154, wherein the population of cells is at least 95%SDC2+. 156. The composition of any one of embodiments 147 to 155,wherein the population of cells is at least 99% SDC2+. 157. Thecomposition of any one of embodiments 147 to 156, wherein the populationof cells is at least 20% CD39+. 158. The composition of any one ofembodiments 147 to 157, wherein the population of cells is at least 30%CD39+. 159. The composition of any one of embodiments 147 to 158,wherein the population of cells is at least 40% CD39+. 160. Thecomposition of any one of embodiments 147 to 159, wherein the populationof cells is at least 50% CD39+. 161. The composition of any one ofembodiments 147 to 160, wherein the population of cells is at least 60%CD39+. 162. The composition of any one of embodiments 147 to 161,wherein the population of cells is at least 70% CD39+. 163. Thecomposition of any one of embodiments 147 to 162, wherein the populationof cells is at least 80% CD39+. 164. The composition of any one ofembodiments 147 to 163, wherein the population of cells is at least 90%CD39+. 165. The composition of any one of embodiments 147 to 164,wherein the population of cells is at least 95% CD39+. 166. Thecomposition of any one of embodiments 147 to 165, wherein the populationof cells is at least 99% CD39+. 167. The composition of any one ofembodiments 147 to 166, wherein at least 90% of the population of cellsis CD45−. 168. The composition of any one of embodiments 147 to 167,further comprising at least 10{circumflex over ( )}6 exosomes isolatedfrom a population of cells bound to a CD39 binding agent. 169. Thecomposition of any one of embodiments 147 to 168, further comprising atleast 10{circumflex over ( )}7 exosomes isolated from a population ofcells bound to a CD39 binding agent. 170. The composition of any one ofembodiments 147 to 169, further comprising at least 10{circumflex over( )}8 exosomes isolated from a population of cells bound to a CD39binding agent. 171. The composition of any one of embodiments 147 to170, further comprising at least 1 mg of exosomes isolated from apopulation of cells bound to a CD39 binding agent. 172. The compositionof any one of embodiments 147 to 171, further comprising at least 10 mgof exosomes isolated from a population of cells bound to a CD39 bindingagent. 173. The composition of any one of embodiments 147 to 172,further comprising at least 20 mg of exosomes isolated from a populationof cells bound to a CD39 binding agent. 174. The composition of any oneof embodiments 147 to 173, further comprising at least 50 mg of exosomesisolated from a population of cells bound to a CD39 binding agent. 175.The composition of any one of embodiments 147 to 174, further comprisingat least 100 mg of exosomes isolated from a population of cells bound toa CD39 binding agent. 176. The composition of any one of embodiments 147to 175, further comprising at least 150 mg of exosomes isolated from apopulation of cells bound to a CD39 binding agent. 177. The compositionof any one of embodiments 147 to 176, further comprising at least 200 mgof exosomes isolated from a population of cells bound to a CD39 bindingagent. 178. The composition of any one of embodiments 147 to 177,further comprising at least 250 mg of exosomes isolated from apopulation of cells bound to a CD39 binding agent. 179. The compositionof any one of embodiments 147 to 178, further comprising at least 500 mgof exosomes isolated from a population of cells bound to a CD39 bindingagent. 180. The composition of any one of embodiments 147 to 179,further comprising at least 750 mg of exosomes isolated from apopulation of cells bound to a CD39 binding agent. 181. The compositionof any one of embodiments 147 to 180, further comprising at least 1000mg of exosomes isolated from a population of cells bound to a CD39binding agent. 182. The composition of any one of embodiments 147 to181, further comprising a buffer. 183. The composition of any one ofembodiments 147 to 182, further comprising a cyropreservant comprisingDMSO. 184. The composition of any one of embodiments 147 to 182, furthercomprising a cyropreservant comprising glycerol. 185. The composition ofany one of embodiments 147 to 184, comprising at least 10{circumflexover ( )}3 cells. 186. The composition of any one of embodiments 147 to185, comprising at least 10{circumflex over ( )}4 cells. 187. Thecomposition of any one of embodiments 147 to 186, comprising at least10{circumflex over ( )}5 cells. 188. The composition of any one ofembodiments 147 to 187, comprising at least 10{circumflex over ( )}6cells. 189. The composition of any one of embodiments 147 to 188,comprising at least 10{circumflex over ( )}7 cells. 190. The compositionof any one of embodiments 147 to 189, wherein the population of cellscomprises CD25+ FoxP3+ regulatory T cells. 191. The composition of anyone of embodiments 147 to 190, wherein the population of cells isgenetically modified to overexpress an apyrase. 192. The composition ofany one of embodiments 147 to 191, wherein the population of cells isgenetically modified to overexpress CD39. 193. The composition of anyone of embodiments 147 to 192, wherein the population of cells isgenetically modified to overexpress CD39L3. 194. A compositioncomprising exosomes, wherein the exosomes are at least 20% SDC2+ andwherein the exosomes are bound to a CD39 binding agent. 195. Thecomposition of embodiment 194, wherein the exosomes are at least 30%SDC2+. 196. The composition of embodiment 194 or embodiment 195, whereinthe exosomes are at least 40% SDC2+. 197. The composition of any one ofembodiments 194 to 196, wherein the exosomes are at least 50% SDC2+.198. The composition of any one of embodiments 194 to 197, wherein theexosomes are at least 60% SDC2+. 199. The composition of any one ofembodiments 194 to 198, wherein the exosomes are at least 70% SDC2+.200. The composition of any one of embodiments 194 to 199, wherein theexosomes are at least 80% SDC2+. 201. The composition of any one ofembodiments 194 to 200, wherein the exosomes are at least 90% SDC2+.202. The composition of any one of embodiments 194 to 201, wherein theexosomes are at least 95% SDC2+. 203. The composition of any one ofembodiments 194 to 202, wherein the exosomes are at least 99% SDC2+.204. The composition of any one of embodiments 194 to 203, wherein theexosomes are at least 20% CD39+. 205. The composition of any one ofembodiments 194 to 204, wherein the exosomes are at least 30% CD39+.206. The composition of any one of embodiments 194 to 205, wherein theexosomes are at least 40% CD39+. 207. The composition of any one ofembodiments 194 to 206, wherein the exosomes are at least 50% CD39+.208. The composition of any one of embodiments 194 to 207, wherein theexosomes are at least 60% CD39+. 209. The composition of any one ofembodiments 194 to 208, wherein the exosomes are at least 70% CD39+.210. The composition of any one of embodiments 194 to 209, wherein theexosomes are at least 80% CD39+. 211. The composition of any one ofembodiments 194 to 210, wherein the exosomes are at least 90% CD39+.212. The composition of any one of embodiments 194 to 211, wherein theexosomes are at least 95% CD39+. 213. The composition of any one ofembodiments 194 to 212, wherein the exosomes are at least 99% CD39+.214. The composition of any one of embodiments 194 to 213, wherein thepopulation is CD45−. 215. The composition of any one of embodiments 194to 214, comprising at least 10{circumflex over ( )}6 exosomes. 216. Thecomposition of any one of embodiments 194 to 215, comprising at least10{circumflex over ( )}7 exosomes. 217. The composition of any one ofembodiments 194 to 216, comprising at least 10{circumflex over ( )}8exosomes. 218. The composition of any one of embodiments 194 to 217,comprising at least 1 μg of exosomes. 219. The composition of any one ofembodiments 194 to 218, comprising at least 10 μg of exosomes. 220. Thecomposition of any one of embodiments 194 to 219, comprising at least 20μg of exosomes. 221. The composition of any one of embodiments 194 to220, comprising at least 50 μg of exosomes. 222. The composition of anyone of embodiments 194 to 221, comprising at least 100 μg of exosomes.223. The composition of any one of embodiments 194 to 222, comprising atleast 150 μg of exosomes. 224. The composition of any one of embodiments194 to 223, comprising at least 200 μg of exosomes. 225. The compositionof any one of embodiments 194 to 224, comprising at least 250 μg ofexosomes. 226. The composition of any one of embodiments 194 to 225,comprising at least 500 μg of exosomes. 227. The composition of any oneof embodiments 194 to 226, comprising at least 750 μg of exosomes. 228.The composition of any one of embodiments 194 to 227, comprising atleast 1000 μg of exosomes. 229. The composition of any one ofembodiments 194 to 228, comprising a buffer.

Definitions

In some cases “exosomes” or “microvesicles” used interchangeably hereininclude cell-derived vesicles, which are released into the extracellularenvironment by a cell, for example a cultured cell or a cultured stromalcell.

“SDC2,” also known as syndecan-2, CD362, S2, or fibroglycan, refersgenerally herein to the SDC2 polypeptide specified by the sequencelisting, or the polypeptide encoded by the SDC2 locus. Syndecan-2, or‘the SDC2 protein’ or simply SDC2, is a transmembrane type I heparinsulfate proteoglycan. Additional synonyms for syndecan-2, aside from‘the SDC2 protein’ or SDC2, include HSPG, CD362, HSPG1, and SYND2.Generally, as used herein SDC2 refers to the protein or a recognizablefragment thereof unless otherwise indicated, for example by reciting‘the SDC2 gene,’ ‘the SDC2 transcript,’ ‘an SDC2 antibody.’Additionally, SDC2 is identified by its polypeptide sequence in thesequence listing that accompanies this specification. An SDC2 fragmentrefers to any set of consecutive residues of SDC2 that uniquely orrecognizably map to the SDC2 polypeptide sequence. In some cases an SDC2fragment retains some or all activity of the SDC2 protein, or acts as aninhibitor of full length or native SDC2. SDC2 also occasionally refersinformally herein to the locus or gene encoding the SDC2 protein. In theevent that one of skill in the art is unable to distinguish an SDC2reference, it is presumed that the term is used herein in reference tothe protein or polypeptide rather than to the gene, transcript, or anantibody raised against or binding to SDC2. There is a family ofsyndecan proteins in mammals. SDC2 is used alternately in reference to amammalian syndecan-2 or to human SDC2 specifically. In the event thatone of skill in the art is unable to distinguish an SDC2 reference, itis presumed that the term is used herein in reference to the humanprotein or polypeptide.

“Extracellular nucleotidases,” also known as “apyrases,” “E-typenucleotidases,” and “NTPases” as used herein are mediators ofextracellular nucleotide catabolism. Examples of extracellularnucleotideases include but are not limited to CD39, CD39L1, CD39L2,CD39L3, CD39L4, and CD73. Extracellular nucleotideases herein, in somecases, are cell surface proteins for isolation of SDC2+ stromal stemcells. Extracellular nucleotideases herein, in some cases, mediatecatabolism of extracellular ATP to adenosine.

“CD39,” also known as ENTPD1, ATPDase, NTPDase-1, SPG64, andectonucleoside triphosphate diphosphohydrolase 1, refers to thepolypeptide encoded by the CD39 locus. Generally, as used herein, CD39refers to the protein or a recognizable fragment thereof unlessotherwise indicated, for example by reciting ‘the CD39 gene,’ ‘the CD39transcript,’ ‘a CD39 antibody.’ CD 39 is a cell surface protein havingan enzyme that catalyses the hydrolysis of γ and β phosphate residues oftriphospho- and diphosphonucleosides to the monophosphonucleosidederivative. In some cases, CD39 hydrolyzes extracellular ATP toextracellular adenosine.

“CD39L3,” also known as ENTPD3, HB6, NTPDase-3, or ectonucleosidetriphosphage diphosphohydrolase 3, refers to the polypeptide encoded bythe CD39L3 locus. Generally, as used herein, CD39L3 refers to theprotein or a recognizable fragment thereof unless otherwise indicated,for example by reciting ‘the CD39L3 gene,’ ‘the CD39L3 transcript,’ ‘aCD39L3 antibody.’ CD 39L3 is a cell surface protein having an enzymethat catalyses the hydrolysis of γ and β phosphate residues oftriphospho- and diphosphonucleosides to the monophosphonucleosidederivative. In some cases, CD39L3 hydrolyzes extracellular ATP toextracellular adenosine.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”,are used interchangeably herein and in some cases, refer to anymammalian subject for whom diagnosis, treatment, or therapy is desired,particularly humans. “Mammal” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and laboratory, zoo, sports, or pet animals, such as dogs,horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guineapigs, monkeys etc. In some embodiments, the mammal is human.

As used herein, the terms “treatment,” “treating,” and the like, in somecases, refer to administering an agent, or carrying out a procedure, forthe purposes of obtaining an effect. The effect may be prophylactic interms of completely or partially preventing a disease or symptom thereofand/or may be therapeutic in terms of effecting a partial or completecure for a disease and/or symptoms of the disease. “Treatment,” as usedherein, may include treatment of a tumor in a mammal, particularly in ahuman, and includes: (a) preventing the disease or a symptom of adisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it (e.g., includingdiseases that may be associated with or caused by a primary disease; (b)inhibiting the disease, i.e., arresting its development; and (c)relieving the disease, i.e., causing regression of the disease. Treatingmay refer to any indicia of success in the treatment or amelioration orprevention of an cancer, including any objective or subjective parametersuch as abatement; remission; diminishing of symptoms or making thedisease condition more tolerable to the patient; slowing in the rate ofdegeneration or decline; or making the final point of degeneration lessdebilitating. The treatment or amelioration of symptoms can be based onobjective or subjective parameters; including the results of anexamination by a physician. Accordingly, the term “treating” includesthe administration of the compounds or agents of the present inventionto prevent or delay, to alleviate, or to arrest or inhibit developmentof the symptoms or conditions associated with cancer or other diseases.The term “therapeutic effect” refers to the reduction, elimination, orprevention of the disease, symptoms of the disease, or side effects ofthe disease in the subject.

The terms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and in some cases,represent that the materials are capable of administration to or upon ahuman without the production of undesirable physiological effects to adegree that would prohibit administration of the composition.

A “therapeutically effective amount” in some cases means the amountthat, when administered to a subject for treating a disease, issufficient to effect treatment for that disease.

As used herein, the term “about” a number refers to a range spanningthat from 10% less than that number through 10% more than that number,and including values within the range such as the number itself.

As used herein, the term “comprising” an element or elements of a claimrefers to those elements but does not preclude the inclusion of anadditional element or elements.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. The present examples, along with the methodsdescribed herein are presently representative of preferred embodiments,are exemplary, and are not intended as limitations on the scope of theinvention. Changes therein and other uses which are encompassed withinthe spirit of the invention as defined by the scope of the claims willoccur to those skilled in the art.

Example 1: Isolation of SDC2+ Stromal Stem Cells from Umbilical Cord

Cells were isolated from human umbilical cord and stromal stem cellswere purified using FACS, gating for live, single cells, CD39 and CD362(SDC2) using a Miltenyi MACSQuant Tyto microchip cell sorter (FIG. 1A,FIG. 1B). The population was analyzed at it was found that thepercentage of the population that is CD362+ was the same as thepercentage of the population that is CD39+/CD362+, 0.10% (FIG. 1C).Colony forming activity was measured on the CD39+/CD362+ cells and itwas found that they formed colonies at a rate of 1/2352 when plated at40k cells per well and a rate of 1/1666 when plated at 10k cells perwell (FIG. 1D). This is compared to unsorted cells which formed coloniesat a rate of 1/9090 when plated at 200k cells per well and a rate of1/9375 when plated at 100k cells per well (FIG. 1E). Therefore, stromalstem cells isolated using CD39 as a marker perform as well or betterthan stromal stem cells isolated using CD362 as a marker.

Example 2: Isolation of SDC2+ Stromal Stem Cells from Bone Marrow

Cells were isolated from human bone marrow and stromal stem cells werepurified using FACS with a BD FACSAria cell sorter isolatingCD39−/CD362+, CD39+/CD362+, and CD39+/CD362− fractions (FIG. 2A). Thepopulations were analyzed and it was found that CD39+/CD362+ cellsaccounted for about 0.4% of the population while CD39 hi/CD362+ cellsaccounted for 0.02% of the population (FIG. 2B). Therefore, most CD362+cells were found to be also CD39+. Sorted cells were further analyzedfor colony formation (FIG. 2C) showing that unsorted bone marrow cells(wild type) form colonies at a rate of 1/18750 to 1/50000; CD39−/CD362+cells form colonies at a rate of 0 to 1/11; CD39+/CD362+ cells formcolonies at a rate of 1/46-1/4; and CD39+/CD362− and double negativecells form colonies at a rate of 0.

In an alternative method, cells were isolated from human bone marrow andstromal stem cells were purified using FACS with a Miltenyi MACSQuantTyto microchip cell sorter isolating CD39−/CD362+, CD39+/CD362+, andCD39+/CD362− fractions (FIG. 3A). Sorted cells were further analyzed forcolony formation comparing results of the BD FACSAria with the MiltenyiMACSQuant Tyto (FIG. 3B) showing that unsorted bone marrow cells (wildtype) form colonies at a rate of 1/50000 to 1/18750 (BD) versus 1/14285(Tyto); CD39−/CD362+ cells form colonies at a rate of 0-1/11 (BD;CD39+/CD362+ cells form colonies at a rate of 1/46 to 1/4 (BD) versus1/16.6 (Tyto); CD39+/CD362− cells form colonies at a rate of 0 (BD); anddouble negative cells form colonies at a rate of 0 (BD) versus 1/100000to 1/16.6 (Tyto).

Example 3: Treatment of Diabetic Ulcers

A therapeutically active CD39+/SDC2+ cell composition is used to treatan individual who is in need of treatment of diabetic ulcers. A doctorapplies the therapeutically active CD39+/SDC2+ cell compositionformulated with a collagen ointment for topical use to the diabeticulcers. One week after administration of the composition to the diabeticulcers, the ulcers have decreased in severity. Optionally, the patientreturns for another administration of the composition. One month afteradministration of the composition to the diabetic ulcers, the ulcershave largely healed.

Example 4: Treatment of Inflammatory Liver Disease

A therapeutically active CD39+/SDC2+ cell composition is used to treatan individual who is in need of treatment of autoimmune hepatitis. Adoctor administers the therapeutically active in vitro SDC2+ cellcomposition formulated for intravenous. After administration of thecomposition to the subject, liver function of the individual improvesand the individual does not require a liver transplant.

Example 5: Scratch Wound Assay. A scratch wound assay is performed onA549 cells to observe the effect of CD39+/SDC2+ cells on cell migration.In this assay, 300,000 A549 cells are grown in a monolayer in a 24 wellplate. The cells are scraped with a p200 pipet tip in a straight line tocreate a scratch. An image is taken at the time of scraping (0 h) andafter a 48 hour incubation with serum free media or 10{circumflex over( )}6 CD39+/SDC2+ cells in serum free media. Incubation with cellsresults in a reduced scratch size compared to serum free media.

This example shows increased cell migration in cells treated withCD39+/SDC2+ cells compared to cells treated with serum free media alone.

Example 6: NFκB Reporter Assay. An NFκB assay is performed on A549 cellstreated with serum free media or CD39+/SDC2+ cells in serum free media.In this assay, 30,000 A549 cells that are transfected with anNFκB-luciferase reporter gene are grown in a 96 well dish. The cells aretreated with serum free media or 10{circumflex over ( )}5 CD39+/SDC2+cells in serum free media for 24 hours and then are stimulated withhuman IL-1β for 24 hours and luciferase activity is measured. Areduction is observed in luciferase activity in A549 cells treated withCD39+/SDC2+.

This example shows a reduction in the inflammatory response in cellstreated with CD39+/SDC2+ cells compared to cells treated with serum freemedia alone.

Example 7: Exosome delivery to treat ARDS lung injury. CD39+/SDC2+ cellsare tested in a rat model of ARDS. Briefly, 300 g Sprague Dawley ratsare given an intrapulmonary dose of E. coli to induce ARDS lung injury,one hour later, the rats are treated with an intravenous dose of 200 μgor 10 mill/kg CD39+/SDC2+ cells. The extent of lung injury is thenmeasured 24 hours later by measurement of arterial oxygen, BAL bacterialload, BAL cell infiltration, static compliance, and a lung wet dryratio. An improvement in arterial FI 0.3 is observed by treatment withCD39+/SDC2+ cells compared to vehicle (one way anova p<0.0001). Animprovement is also observed in arterial FI 1.0 observed by treatmentwith CD39+/SDC2+ cells compared to vehicle (one way anova p<0.0001).This demonstrates that CD39+/SDC2+ cells are able to treat lung damagein an ARDS lung injury model.

Static lung compliance is also measured in rats treated with CD39+/SDC2+cells compared to PBS vehicle control. This measurement showsCD39+/SDC2+ cells effective in treating ARDS lung injury compared tocontrol (one way anova p<0.01).

Bacterial load is measured in the bronchioalveolar lavage (BAL). In thismeasurement, a significant reduction is observed in BAL bacterial loadin rats treated with CD39+/SDC2+ cells (one way anova p<0.01). In thisassay, a significant difference is observed in BAL total cell count andBAL neutrophil count in rats treated with CD39+/SDC2+ cells compared tovehicle control (one way anova p<0.01).

This example shows CD39+/SDC2+ cells administration with an increaseddose and intravenous mode of administration treats ARDS lung injury.

Example 8: Treatment of Diabetes-Associated Kidney Failure

A therapeutically active CD39+/SDC2+ cells composition is used to treatan individual suffering from kidney failure related to diabetes. Afteradministration of the composition to the subject, the symptomsexperienced by the patient are decreased. When 10 patients are given theintravenous injections of the composition, 9 experience marked increasein kidney function. When 10 patients are given an alternative treatment,the kidney function is not improved. No impact upon blood glucose levelsis observed in some individuals.

Example 9: In Vitro Exosomes. A therapeutically active composition isprepared comprising in vitro SDC2+ exosomes. The SDC2+ exosomecomposition is prepared to include infliximab contained within theexosome composition. When the composition is administered to thepatient, the infliximab is not exposed to the humoral immune system andthe patient does not develop humoral immune response toward theinfliximab. When a sample of the composition is tested, at least 30% ofthe exosomes are found to comprise SDC2. The exosome composition isfrozen for storage in phosphate buffer alone without the use of acryoprotectant such as DMSO. When the composition is thawed, there is noloss therapeutic efficacy as measured by inhibition of the inflammatoryresponse.

Example 10: In Vitro Exosomes and SDC2+ Mesenchymal Stromal Stem Cells.A therapeutically active composition is prepared comprising in vitroSDC2+ exosomes. The SDC2+ exosome composition is prepared to includeinfliximab contained within the exosome composition. When thecomposition is administered to the patient, the infliximab is notexposed to the humoral immune system and the patient does not develophumoral immune response toward the infliximab. When a sample of thecomposition is tested, at least 30% of the exosomes are found tocomprise SDC2. The exosome composition is combined with SDC2+mesenchymal stromal stem cells, purified based on expression of CD39.Addition of the exosome composition enhances the therapeutic activity ofthe SDC2+ mesenchymal stromal stem cells in reducing the inflammatoryresponse.

Example 11: In Vitro Exosomes and Regulatory T Cells. A therapeuticallyactive composition is prepared comprising in vitro SDC2+ exosomes fromcells isolated based on expression of CD39. The SDC2+ exosomecomposition is prepared to include infliximab contained within theexosome composition. When the composition is administered to thepatient, the infliximab is not exposed to the humoral immune system andthe patient does not develop humoral immune response toward theinfliximab. When a sample of the composition is tested, at least 30% ofthe exosomes are found to comprise SDC2. The exosome composition iscombined with CD25+CD4+Foxp3+ regulatory T cells. Addition of theexosome composition enhances the therapeutic activity of theCD25+CD4+Foxp3+ regulatory T cells in reducing the inflammatoryresponse.

Example 12: Treatment of Diabetic Ulcers. A therapeutically active invitro SDC2+ exosome composition is used to treat an individual who is inneed of treatment of diabetic ulcers. A doctor prescribes thetherapeutically active in vitro SDC2+ exosome composition formulatedwith a collagen ointment for topical use and instructs the patient toadminister the composition 1-5 times per week to the diabetic ulcers.After administration of the composition to the diabetic ulcers for oneweek, the ulcers have decreased in severity. After administration of thecomposition to the diabetic ulcers for one month, the ulcers havelargely healed.

Example 13: Treatment of Rheumatoid Arthritis. A therapeutically activein vitro SDC2+ exosome composition additionally comprising infliximab isused to treat an individual who is in need of treatment of rheumatoidarthritis. A doctor prescribes the therapeutically active in vitro SDC2+exosome composition formulated for subcutaneous administration andinstructs the patient to administer the composition weekly bysubcutaneous injection. After administration of the composition to thesubject, the joint pain experienced by the patient is decreased by atleast 50% and the joint mobility of the patient is increased by at least60%. Further, the patient does not experience a humoral immune responseto infliximab.

Example 14: Treatment of Amyotrophic Lateral Sclerosis. Atherapeutically active in vitro SDC2+ exosome composition is used totreat an individual who is in need of treatment of amyotrophic lateralsclerosis (ALS). A doctor prescribes the therapeutically active in vitroSDC2+ exosome composition formulated for intrathecal administration atthe doctor's office and instructs the patient to come for weeklyintrathecal injections. After administration of the composition to thesubject, the symptoms experienced by the patient are decreased. When 10patients are given the intrathecal injections of the composition, 8 ofthem experience minimal decrease in motor neuron function andstabilization of their condition. When 10 patients are given analternative treatment, 3 of them experience minimal decrease minimaldecrease in motor neuron function and stabilization of their condition.

Example 15: Isolation. An immuno-modulatory composition is obtained byisolating exosomes that express SDC2. The composition is isolated fromSDC2+ stromal cells that have been transformed with a lentivirus thatcarries the SDC2 gene such that the SDC2+ stromal cells areover-expressing SDC2. Over-expression of SDC2 has the effect ofincreasing the number of SDC2+ exosomes and as a consequence increasesthe efficiency of isolating the exosomes and the potency of the exosomesproduced by the cells.

The isolated exosomes are analyzed and it is determined that at least20% of the exosomes are SDC2+ and 700 mg of exosomes are obtained. Theexosomes are diluted using a phosphate buffer and frozen without DMSO insingle use vials until needed for use.

Example 16: Isolation Using Antibody Purification. An immuno-modulatorycomposition is obtained by isolating exosomes that express SDC2. Thecomposition is isolated from SDC2+ stromal cells that have beentransformed with a lentivirus or adenovirus that carries the SDC2 genesuch that the SDC2+ stromal cells are over-expressing SDC2.Over-expression of SDC2 has the effect of increasing the number of SDC2+exosomes and as a consequence increases the efficiency of isolating theexosomes. The composition comprising exosomes is incubated with ananti-SDC2 antibody for 10-30 minutes or overnight at 4° C. The SDC2+exosomes are isolated from the solution using flow cytometry basedsorting or column chromatography and the SDC2+ exosomes are eluted fromthe column.

The isolated exosomes are analyzed and it is determined that at least80% of the exosomes are SDC2+ and 700 mg of exosomes are obtained. Theexosomes are diluted using a phosphate buffer and frozen without DMSO insingle use vials until needed for use.

Example 17: Isolation Using Ultracentrifugation. An immuno-modulatorycomposition is obtained by isolating exosomes that express SDC2. Thecomposition is isolated from SDC2+ stromal cells that have beentransformed with a lentivirus or adenovirus that carries the SDC2 genesuch that the SDC2+ stromal cells are over-expressing SDC2.Over-expression of SDC2 has the effect of increasing the number of SDC2+exosomes and as a consequence increases the efficiency of isolating theexosomes. Supernatant from the SDC2+ cells containing the exosomes issubjected to ultracentrifugation at 100,000×g force for 16 hours at 4°C. The exosome fraction is isolated from the media solution. For someuses, ultrafiltration with a subsequent liquid chromatography (UF-LC)steps is performed on the resulting product to produce a more purepreparation of SDC2+ exosomes.

The isolated exosomes are analyzed and it is determined that at least80% of the exosomes are SDC2+ and 700 mg of exosomes are obtained. Theexosomes are diluted using a phosphate buffer and frozen without DMSO insingle use vials until needed for use.

Example 18: Isolation Using Hollow-Fiber Bioreactors (HFBRs). Humanmesenchymal stem cells (MSCs), such as SDC2+ MSCs are grown in a C2011cartridge (FiberCell Systems), or alternatively in a Terumo Quantum CellExpansion System, seeded with 1×10⁷ to 1×10⁸ MSCs. It continuouslyproduces exosomes for 10 weeks. The typical harvest from theextracapillary space of the cartridge is 1.1×10¹² exosomes/ml in avolume of 400 ml. The total bioreactor yield or exosomes by number isapproximately 10-fold higher at a concentration that was 10-fold higher.Harvests are performed every two weeks, and prior to each harvest, smallsamples of cells are collected for phenotypic analysis. During these 10weeks, the culture does not expand based on glucose uptake rate, whichremains fairly constant. By orthogonal measure, the phenotype of thecells remains constant as well. Exosomes are continuously harvestedwithout splitting and/or subculturing of the cells.

Example 19: Delivery of an Exosome Composition. An immuno-modulatorysignal is delivered to the intracellular space of an individual usingexosomes isolated from SDC2+ stem cells. The exosomes are formulated tocontain infliximab. The exosomes are administered to the individualsubcutaneously and the contents of the exosome are delivered to theintracellular space of the individual, thereby delivering theimmuno-modulatory signal. Administration of the exosomes and delivery ofthe immuno-modulatory signal does not result in a humoral immuneresponse by the individual to the infliximab, thereby increasing thepotency of the infliximab.

Example 20: Supplemented Stem Cell Compositions. A therapeuticallyactive composition is prepared comprising in vitro SDC2+ exosomesderived from mesenchymal stromal stem cells. When a sample of theprepared composition is tested, at least 30% of the exosomes are foundto comprise SDC2. The SDC2+ exosomes are combined with the mesenchymalstromal stem cells and the resulting mixture is administered to anindividual in need of wound healing. The result of this treatment isenhanced wound healing compared to administration of the mesenchymalstromal stem cells alone.

Example 21: Exosome delivery to treat ARDS lung injury. Exosomes weretested in a rat model of ARDS. Briefly, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated with a 90-100 μg dose of exosomes or 4million human mesenchymal stem cells (hMSC). The extent of lung injurywas then measured 24 hours later by measurement of arterial oxygen, BALbacterial load, BAL cell infiltration, static compliance, and a lung wetdry ratio. An improvement in arterial FI 0.3 was observed by treatmentwith exosomes compared to vehicle (FIG. 7). This demonstrates thatexosomes are able to treat lung damage in an ARDS lung injury model.Exosome treatment was shown to be safe as PBS vehicle treatment inarterial FI 1.0 measurement which showed a similar measurement betweenexosome treatment and PBS vehicle control (FIG. 8).

Arterial oxygen was also measured for bone marrow (BM) exosomes andhuman umbilical cord (HUC) exosome treatment with arterial FI 0.3 (FIG.9) and arterial FI 1.0 (FIG. 10) compared to PBS vehicle control. InFIG. 9 and FIG. 10, BM exosomes and HUC exosomes were shown to be justas safe as PBS vehicle control on arterial FI 0.3 or FI 1.0.

In FIG. 11 bacterial load was measured in the bronchioalveolar lavage(BAL). In this measurement, a measurable reduction was observed in BALbacterial load in rats treated with exosomes and hMSC. BAL total cellcount and neutrophil count are shown in FIG. 12 and FIG. 13. In thisassay, a measurable difference was observed in BAL total cell count andBAL neutrophil count in rats treated with exosomes and hMSC compared tovehicle control.

Static lung compliance and lung wet dry ratio were also measured in ratstreated with exosomes and hMSC compared to PBS vehicle control (FIG.14). This measurement showed exosomes were just as safe as PBS vehiclecontrol. FIG. 15 shows the results of wet dry ratio in rats that weretreated with exosomes or hMSC. This also showed that exosomes were justas safe as PBS vehicle control.

This example shows safety and efficacy of exosomes compared to PBSvehicle control.

Example 22: Scratch Wound Assay. A scratch wound assay was performed onA549 cells to observe the effect of exosomes on cell migration. In thisassay, 300,000 A549 cells were grown in a monolayer in a 24 well plate.The cells were scraped with a p200 pipet tip in a straight line tocreate a scratch. An image was taken at the time of scraping (0 h) andafter a 48 hour incubation with serum free media or 2 μg SDC2+ exosomesin serum free media. As shown in FIG. 16 and FIG. 17, incubation withexosomes resulted in a reduced scratch size compared to serum freemedia.

This example shows increased cell migration in cells treated withexosomes compared to cells treated with serum free media alone.

Example 23: NFκB Reporter Assay. An NFκB assay was performed on A549cells treated with serum free media or exosomes in serum free media. Theresults of these assays are shown in FIGS. 18-21. In this assay, 30,000A549 cells that were transfected with an NFκB-luciferase reporter genewere grown in a 96 well dish. The cells were treated with serum freemedia or 2 μg exosomes in serum free media for 24 hours and thenstimulated with human IL-10 for 24 hours and luciferase activity wasmeasured. A reduction was observed in luciferase activity in A549 cellstreated with exosomes obtained by ultrafiltration followed byultracentrifugation (FIG. 18 and FIG. 10) as well as exosomes obtainedby ultracentrifugation without ultrafiltration (FIG. 20 and FIG. 21).

This example shows a reduction in the inflammatory response in cellstreated with exosomes compared to cells treated with serum free mediaalone.

Example 24: Exosome delivery to treat ARDS lung injury. Exosomes weretested in a rat model of ARDS. Briefly, 300 g Sprague Dawley rats weregiven an intrapulmonary dose of E. coli to induce ARDS lung injury, onehour later, the rats were treated with an intravenous dose of 200 μg or10 mill/kg human mesenchymal stem cells (hMSC). The extent of lunginjury was then measured 24 hours later by measurement of arterialoxygen, BAL bacterial load, BAL cell infiltration, static compliance,and a lung wet dry ratio. An improvement in arterial FI 0.3 was observedby treatment with exosomes compared to vehicle (one way anova p<0.0001)(FIG. 22). An improvement was also observed in arterial FI 1.0 wasobserved by treatment with exosomes compared to vehicle (one way anovap<0.0001) (FIG. 23). This demonstrates that exosomes are able to treatlung damage in an ARDS lung injury model.

Static lung compliance was also measured in rats treated with exosomesor hMSC compared to PBS vehicle control (FIG. 24). This measurementshowed exosomes effective in treating ARDS lung injury compared tocontrol (one way anova p<0.01).

In FIG. 25 bacterial load was measured in the bronchioalveolar lavage(BAL). In this measurement, a significant reduction was observed in BALbacterial load in rats treated with exosomes and hMSC (one way anovap<0.01). BAL total cell count and neutrophil count are shown in FIG. 26and FIG. 27. In this assay, a significant difference was observed in BALtotal cell count and BAL neutrophil count in rats treated with exosomesand hMSC compared to vehicle control (one way anova p<0.01).

This example shows exosome administration with an increased dose andintravenous mode of administration treats ARDS lung injury.

Example 25: Treatment of Diabetes-Associated Kidney Failure

A therapeutically active in vitro SDC2+ exosome composition is used totreat an individual suffering from kidney failure related to diabetes.After administration of the composition to the subject, the symptomsexperienced by the patient are decreased. When 10 patients are given theintrathecal injections of the composition, 9 experience marked increasein kidney function. When 10 patients are given an alternative treatment,the kidney function is not improved. No impact upon blood glucose levelsis observed in some individuals.

Example 26: Treatment of Alzheimer's Disease

A therapeutically active in vitro SDC2+ exosome composition is used totreat an individual suffering from Alzheimer's disease. Afteradministration of the composition to the subject by intravenousadministration, the composition is able to cross the blood brain barrierand treat the brain and Alzheimer's disease symptoms are decreased. When10 patients are given the intravenous injection of the composition, 9experience an improvement in memory.

Example 27: Improved SDC2+ Stromal Stem Cells

A SDC2+ stromal stem cell composition is isolated from a human umbilicalcord using a CD39 antibody. The composition further comprises CD25+FoxP3+ regulatory T cells. One third of the cells are transduced with alentivirus to overexpress CD39, one third of the cells are transducedwith a lentivirus to overexpress CD39L3, and one third of the cells aretransduced with a lentivirus with no transgene. The SDC2+ stromal stemcell compositions transduced with CD39 or CD39L3 are more effective ininhibition an inflammatory response as measured in a NFκB reporter assaythan SDC2+ stromal stem cell compositions transduced with a lentiviruswith no transgene. This example shows that SDC2+ stromal stem cellcompositions have greater anti-inflammatory activity when transducedwith CD39 or CD39L3.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-11. (canceled)
 12. A method of modulating an inflammation response ina mammal comprising delivering a composition comprising SDC2+ cells to asite of the inflammation response, wherein the SDC2+ cells are isolatedfrom a mixed population of cells by isolating cells based upon CD39expression.
 13. The method of claim 12, wherein isolating cellscomprises collecting cells bound to a CD39 binding agent.
 14. The methodof claim 13, wherein isolating the cells comprises culturing the cellsbound to the CD39 binding agent.
 15. The method of claim 12, whereindelivering comprises injecting the composition comprising SDC2+ cells.16. The method of claim 12, wherein delivering comprises topicallyapplying the composition comprising SDC2+ cells.
 17. The method of claim16, wherein the composition comprises a hydrogel.
 18. The method ofclaim 16, wherein the composition comprises a collagen gel.
 19. Themethod of claim 12, wherein delivering comprises intravenous deliverythe composition comprising SDC2+ cells.
 20. The method of claim 12,wherein the CD39 binding agent comprises an antibody.
 21. The method ofclaim 20, wherein the antibody is conjugated to a fluorophore.
 22. Themethod of claim 20, wherein the antibody is conjugated to a bead. 23.The method of claim 12, wherein at least 20% of the SDC2+ cells areSDC2+.
 24. The method of claim 12, wherein at least 40% of the SDC2+cells are SDC2+.
 25. The method of claim 12, wherein at least 70% of theSDC2+ cells are SDC2+.
 26. The method of claim 12, wherein the methodcomprises delivering at least 10{circumflex over ( )}3 SDC2+ cells. 27.The method of claim 12, wherein the inflammation response comprises atleast one of type 1 diabetes, type 2 diabetes, sepsis, Crohn's disease,inflammatory bowel syndrome, rheumatoid arthritis, graft versus hostdisease, multiple sclerosis, ALS, a dermal wound, a bone fracture, aconcussion wound, a burn, atherosclerosis, nephropathy, cardiomyopathy,neuropathy, a kidney disorder, kidney failure, a diabetic ulcer, a legulcer, ARDS, sepsis, inflammatory liver disease, myocarditis,postmyocardial infarction syndrome, postpericardiotomy syndrome,subacute bacterial endocarditis, anti-glomulerular basement membranenephritis, interstitial cystitis, lupus enphritis, autoimmune hepatitis,primary biliary cirrhosis, primary sclerosing cholangitis,antisynthetase syndrome, asthma, chronic obstructive pulmonary disease,cystic fibrosis, atelectasis, bronchitis, emphasema, pneumonia,pulmonary endema, Alopecia Areata, autoimmune Angioedema, autoimmuneprogesterone dermatitis, autoimmune urticaria, Bullous pemphigoid,Cicatricial pemphigoid, Dermatitis herpetiformis, Discoid lupuserythematosus, Epidermolysis bullosa acquisita, Erythema nodosum,Gestational pemphigoid, Hidradenitis suppurativa, Lichen planus, Lichensclerosus, Linear IgA disease, Morphea, Pemphigus vulgaris, Pityriasislichenoides et varioliformis acuta, Mucha-Habermann disease, Psoriasis,Systemic scleroderma, Vitiligo, Addison's disease, autoimmunepolyendocrine syndrome, autoimmune polyendocrine syndrome type 2,autoimmune polyendocrine syndrome type 3, autoimmune pancreatitis,autoimmune thyroiditis, Ord's thyroiditis, Graves' disease, ReproductiveOrgan disorder, autoimmune oophoritis, Endometriosis, autoimmuneorchitis, Sjogren's syndrome, autoimmune enteropathy, Celiac disease,Microscopic colitis, Ulcerative colitis, Antiphospholipid syndrome,Aplastic anemia, autoimmune hemolytic anemia, autoimmunelymphoproliferative syndrome, autoimmune neutropenia, autoimmunethrombocytopenic purpura, Cold agglutinin disease, Essential mixedcryoglobulinemia, Evans syndrome, IgG4-related systemic disease,Paroxysmal nocturnal hemoglobinuria, Pernicious anemia, Pure red cellaplasia, Thrombocytopenia, Adiposis dolorosa, Adult-onset Still'sdisease, Ankylosing Spondylitis, CREST syndrome, Drug-induced lupus,Enthesitis-related arthritis, Eosinophilic fasciitis, Felty syndrome,Juvenile Arthritis, Lyme disease (Chronic), Mixed connective tissuedisease, Palindromic rheumatism, Parry Romberg syndrome,Parsonage-Turner syndrome, Psoriatic arthritis, Reactive arthritis,Relapsing polychondritis, Retroperitoneal fibrosis, Rheumatic fever,Rheumatoid arthritis, Sarcoidosis, Schnitzler syndrome, Systemic LupusErythematosus, Undifferentiated connective tissue disease, cachexia,sarcophenia, Dermatomyositis, Fibromyalgia, Inclusion body myositis,Myositis, Myasthenia gravis, Neuromyotonia, Paraneoplastic cerebellardegeneration, Polymyositis, Acute disseminated encephalomyelitis, Acutemotor axonal neuropathy, Anti-N-Methyl-D-Aspartate ReceptorEncephalitis, Balo concentric sclerosis, Bickerstaffs encephalitis,Chronic inflammatory demyelinating polyneuropathy, Guillain-Barrésyndrome, Hashimoto's encephalopathy, Idiopathic inflammatorydemyelinating diseases, Lambert-Eaton myasthenic syndrome, Multiplesclerosis, Pediatric Autoimmune Neuropsychiatric Disorder Associatedwith Streptococcus, Progressive inflammatory neuropathy, Restless legsyndrome, Stiff person syndrome, Sydenham chorea, amyotrophic lateralsclerosis, Parkinson's disease, Alzheimer's disease, Transversemyelitis, autoimmune retinopathy, autoimmune uveitis, Cogan syndrome,Graves ophthalopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, autoimmune inner ear disease, Meniere's disease,Anti-neutrophil cytoplasmic antibody-associated vasculitis, Behçet'sdisease, Churg-Strauss syndrome, Giant cell arteritis, Henoch-Schonleinpurpura, Kawasaki's disease, Leukocytoclastic vasculitis, Lupusvasculitis, Rheumatoid vasculitis, Microscopic polyangiitis,Polyarteritis nodosa, Polymyalgia rheumatica, Urticarial vasculitis, andVasculitis. 28.-30. (canceled)